CN104250301A

CN104250301A - Engineered antibody constant domain molecules

Info

Publication number: CN104250301A
Application number: CN201410422052.8A
Authority: CN
Inventors: 迪米特尔·S·迪米特罗夫
Original assignee: Goverment Of United States, AS REPRESENTED BY SECRETARY D
Current assignee: Goverment Of United States, AS REPRESENTED BY SECRETARY D; US Department of Health and Human Services
Priority date: 2008-01-31
Filing date: 2009-01-30
Publication date: 2014-12-31
Also published as: EP2250196A2; CA2713281A1; JP5889533B2; US8580927B2; JP2011514149A; JP2014155503A; CN101977932A; AU2009212747B2; US9527903B2; US10358481B2; AU2009212747A1; CN101977932B; CA2713281C; US20100316641A1; WO2009099961A2; EP2594584B1; EP2594584A1; EP2250196B1; US20170081393A1; CA3052615A1

Abstract

Described herein are engineered antibody constant domain molecules, such as CH2 or CH3 domain molecules, comprising at least one mutation, or comprising at least one complementarity determining region (CDR), or a functional fragment thereof, engrafted in a loop region of the CH2 domain. The CH2 domain molecules described herein are small, stable, soluble, exhibit little to no toxicity and are capable of binding antigen.

Description

Engineered antibody constant domain molecules

The divisional application that the application was international filing date on January 30th, 2009, international application no PCT/US2009/032692 enters National Phase in China, application number 200980110221.1, denomination of invention " engineered antibody constant domain molecules " on September 21st, 2010.

The cross reference of related application

This application claims the U.S. Provisional Application the 61/063rd enjoyed and submitting on January 31st, 2008, the right of priority of No. 245, by reference its entirety is incorporated to herein.

Technical field

The present invention relates to antibody, particularly specific position sudden change antibody constant structural domain and/or move into the antibody constant structural domain of one or more variable chain link from the heterologous antibody of specific binding object antigen.

Background technology

Conventional antibody is the large-scale multimeric protein mixture comprising at least four polypeptide chains, and described four polypeptide chains comprise two light chains and two heavy chains.The heavy chain of antibody and light chain contain variable (V) district of conjugated antigen and provide constant (C) district of support structure and effector function.Antigen binding domain comprises two structural domains be separated, heavy-chain variable domains (V _h) and light variable domains (V _l).Complementary determining region (CDR) is the short amino acid sequence in antibody variable territory, and it provides antigen-specific.The heavy chain of antibody molecule and light chain respectively provide three CDR (CDR1, CDR2 and CDR3), therefore, it is possible to have six CDR with each antibody of antigen contact, produce antigen-specific.

Typical antibody such as IgG molecule has the molecular weight of about 150kD.Treatment use may be limited, because the relatively large size of antibody can limit tissue infiltration or epi-position enters.

The many less Fab (such as, Fab, Fab' and F (ab ') of naturally occurring antibody after identifying protease digestion ₂).These antibody fragments have the molecular weight of scope at about 50kD to 100kD.Used the method for restructuring to produce alternative Fab, be called single chain variable fragment (scFv), it is the V engaged by the peptide linker by synthesis _land V _hform.ScFv molecule has the molecular weight of about 25-30kD.

Be made up of a pair variable region although the antigen-binding unit of the naturally occurring antibody in the mankind and other Mammals of great majority is generally known, Camelidae (camelid) species express global function, the antibody with high specificity of the shortage sequence of light chain of vast scale.Camelidae heavy chain antibody as the single heavy chain by its constant region dimerization homodimer and there are (United States Patent (USP) the 5th, 840, No. 526 and the 6th, 838, No. 254; And U.S. Patent Application Publication No. 2003-0088074).The variable domains of these Camelidae heavy chain antibody is called V _hh structure territory, they are being separated into V _hability (the people .Nature 363:446-448 such as Hamers-Casterman, 1993 of high specific conjugated antigen are remained during the fragment of chain; The people .FEBS Lett.414:521-526 such as Gahroudi, 1997).

Single V of conjugated antigen _hstructural domain is called domain antibodies (dAb), also by its mouse V from the genomic DNA amplification by immune mouse _hidentify out in gene library (Ward etc., Nature 341:544-546,1989).Have also been described can the single immunoglobulin variable domain domain polypeptide of the mankind (see such as, PCT publication number WO 2005/035572 and WO 2003/002609) of high-affinity conjugated antigen.

But, to can the demand of very little antibody of specific binding antigen still exist.This micromolecular may provide the epi-position of raising to enter, better tissue infiltration, and may be used to utilize any diagnostic of antibody or its fragment or curative application.

Summary of the invention

The disclosure relates to engineered antibody constant domain molecules.In one embodiment, antibody constant structural domain is the CH2 structural domain from IgG, IgA or IgD.In another embodiment, antibody constant structural domain is the CH3 structural domain from IgE or IgM.As described herein, CH2 or CH3 structural domain molecule is little, stable, solvable, has minimum toxicity and even does not have toxicity and conjugated antigen effectively.Therefore, there is provided herein the polypeptide comprising immunoglobulin (Ig) CH2 or CH3 structural domain, at least one of the ring of wherein said CH2 or CH3 structural domain is suddenlyd change, or being replaced by from the complementary determining region (CDR) of heterologous immunoglobulin variable domains or its function fragment (function fragment as containing specificity determining residue (SDR)) at least partially of the ring region of described CH2 or CH3 structural domain, or the two has concurrently.CH2 and CH3 structural domain molecule as herein described has the molecular weight being less than about 15kD.Additionally provide herein comprise CH2 or CH3 structural domain molecule composition, library and test kit and using method.Additionally provide the restructuring constant domain of the stability showing raising, it can be used as the support building CH2 or the CH3 structural domain that antigen combines.Additionally provide the recombinant C H2 of qualification specific binding antigen or the method for CH3 structural domain and produce the method comprising the library of recombinant C H2 or CH3 structural domain.

The above-mentioned feature and advantage with other become more obvious by the detailed description from following some embodiments of carrying out about accompanying drawing.

Accompanying drawing explanation

Figure 1A is the schematic diagram of immunoglobulin molecules.Conventional antibody is the large-scale multimeric protein mixture comprising at least four polypeptide chains, and described four polypeptide chains comprise two light (L) chains and two weight (H) chains.The heavy chain of antibody and light chain contain variable (V) district of conjugated antigen and provide constant (C) district (as CH1, CH2 and CH3 structural domain) of support structure and effector function.Antigen binding domain comprises two structural domains be separated, heavy-chain variable domains (V _h) and light variable domains (V _l).

Figure 1B shows the consensus amino acid sequences (SEQ ID NO:1) of human heavy chain variable structural domain.Indicate the position of CDR1, CDR2, CDR3 (being expressed as H1, H2 and H3).Also show the aminoacid sequence (SEQ ID NO:2-4) of the heavy chain of three kinds of different Antigen-specific Human's antibody.The numeral of display is based on Kabat numbering system (Wu and Kabat, J.Exp.Med.132 (2): 211-250,1970).

Fig. 2 shows the aminoacid sequence (SEQ ID NO:5) of mankind γ 1CH2 structural domain.Indicate beta sheet (...) and alpha-helix (* * *) region in residue.Also show the position of ring B-C (being expressed as ring 1 here), ring D-E (being expressed as ring 2 here), ring F-G (being expressed as ring 3 here), ring A-B, ring C-D and ring E-F.Residue in each ring shows with runic.

Fig. 3 A-3C is the schematic diagram illustrating the possibility strategy transplanting CDR (or Gao Bianhuan) on CH2 structural domain.

Fig. 4 shows the gel images of the protein expression representing through engineering approaches CH2 structural domain, and described through engineering approaches CH2 structural domain is pointed out by arrow.

Fig. 5 A shows mankind CH2 (NCB accession number J00228; SEQ ID NO:5) and mouse CH2 (NCB accession number J00453; SEQ ID NO:92) amino acid alignment.Identical with similar residue is 67% and 92% respectively.Fig. 5 B is the figure of the size exclusion chromatography of display mankind CH2.Illustration display typical curve.Fig. 5 C is the image of SDS-PAGE gel of molecular weight of display CH2 structural domain molecule (concentration with every swimming lane 1-10 μ g or 2-5 μ g), single chain variable fragment (scFv), antibody fragment (Fab) and complete antibody molecule (IgG).

Fig. 6 A-6B is the figure of the stability of the mankind CH2 that display is measured by circular dichroism (CD) and differential scanning calorimetry (DSC).(A) measured by CD, the folding curve (-) of 25 DEG C, in the unfoldings of 90 DEG C () with the refolding (---) of 25 DEG C.The folding fraction (ff) of albumen is calculated as ff=([θ]-[θ _m])/([θ _t]-[θ _m]).[θ _t] and [θ _m] be wherein 216 at the mean residue ellipticity of the folded state of 25 DEG C and the unfolded state of 90 DEG C.The accurate T from CD is determined from the first order derivative [d (folding fraction)/dT] to temperature (T) _mvalue (54.1 ± 1.2 DEG C).(B) from the thermoinducible unfolding curve of DSC.T _m=55.4 DEG C, its with from the T of CD _msimilar.

Fig. 7 is the schematic diagram of display based on the design of m01 and m02 of CH2 structure.Two C in two natural Cys ^αdistance between s is these two Cys residues define natural disulphide bonds (being pointed out by black arrow).Through engineering approaches disulfide linkage is introduced between V10 and the K104 replaced by halfcystine (m01) or between L12 and K104 (m02).

Fig. 8 is the image of the SDS-PAGE gel of display m01 and m02 high level expression.The solubility expression of m01 and m02 solubility expression and CH2 is contrasted.Expression is pointed out by arrow.

Fig. 9 A-9E is the figure of the stability of the raising of two kinds of mutant that display is measured by CD (A-C), DSC (D) and spectrophotofluorimetry (E).Show m01 (A) and m02 (B) the folding curve (-) of 25 DEG C, in the unfoldings of 90 DEG C () with the refolding (---) of 25 DEG C.(C) the folding fraction of m01 and m02 is calculated by the method identical with CH2.The T of m01 _m=77.4 ± 1.7 DEG C, the T of m02 _m=68.6 ± 0.6 DEG C.(D) the thermoinducible unfolding curve of m01 and m02 is also by DSC record.Compared with CH2, the T of m01 _mwith the T of m02 _mimprove about 20 DEG C and 10 DEG C respectively.(E) by the unfolding of the urea induction of spectrophotofluorimetry contrast between CH2, m01 and m02.The mid point of the unfolding of CH2, m01 and m02 is 4.2M, 6.8M and 5.8M respectively.

Figure 10 A shows the size exclusion chromatography of m01 and m02.As CH2, m01 only form monomer, and m02 mainly forms monomer and form dimer in less degree.Figure 10 B is the figure that display N holds the high stability of CH2 (CH2) and the truncate m01 (m01) cut level with both hands.The first seven N holds residue to be lacked (the residue 1-7 of SEQ ID NO:5).50% unfolding temperature (the T measured by CD _m) (being respectively 62 DEG C and 79 DEG C) significantly high (high 8 DEG C and 5 DEG C respectively) be in the unfolding temperature (being respectively 54 DEG C and 74 DEG C) of corresponding CH2 and m01.

Figure 11 is the schematic diagram of the design in display CH2 library.Show the diagram of CH2 fragment, wherein Filled Rectangle represents ring (L1-L3).Shaded rectangle representative is towards the reverse direction ring (L) from ring 1 to ring 3 and spiral (H1, H2).The empty rectangle representative marked with alphabetical A-G forms seven β chains of β sandwich structure.The initial residue of numeral 231 and 341 representative CH2 fragment when IgG1 and terminal residue.The sequence of CH2 ring 1 (SEQ ID NO:93) and ring 3 (SEQ ID NO:95) illustrates below, and underscore of annotating.The sudden change introduced is presented at (SEQ ID NO:94 and SEQ ID NO:96) in bracket.

Figure 12 A-12B shows the sign of CH2 binding substances (binder).(A) four Bal gp120-CD4 specific C H2 clones are expressed and purifying as described in the following Examples.The product of purifying passes through western blot analysis.Sample 1-4 represents clone m1a1 to the m1a3 ' from solvable fraction, and 5-8 renaturation from inclusion body.(B) elisa assay of the combination of CH2 clone and Bal gp120-CD4.

Figure 13 A-13B is figure and the image of the running gel of the determinant of display CH2 specific binding.(A) ring 1 determines binding ability.Two prevalent clone m1a1 and m1a2 and containing two heterozygotes of nonprimitive CH2 ring 3 sequence are expressed and purifying from inclusion body from ring 1 sequence of m1a1 and m1a3, and be refolded (left figure).Then these albumen are used in (right figure) in elisa assay.(B) CH2 provides critical structures support for ring 1.Carry the prevalent clone m1a1 of extra disulfide linkage and mutant is expressed, purifying and refolding (left figure).Then they are used in (right figure) in elisa assay.

Figure 14 is the extensive neutralization carried out is infected in display to HIV Env pseudotype virus figure by CH2 binding substances.Two CH2 clone m1a1 and m1a2 is used for testing its anti-one group of nine kinds of HIV pseudoviruss neutralising capacity with the fixed concentration of 100 μ g/ml.Concentration is that the C34 peptide of 4 μ g/ml or 6 μ g/ml is used as positive control.

Figure 15 display is based on the design in the 2nd CH2 library of m1a1.The ring 2 (SEQ ID NO:97) of m1a1 and ring 3 (SEQ ID NO:99) sequence (lining out below) is cloned by sequence (SEQ ID NO:98 and the SEQ ID NO:100) replacement shown in bracket from CH2.

Figure 16 A-16D shows the sign of the CH2 clone being selected from the 2nd CH2 library.(A) expression and purification of the CH2 clone in the second library is selected from.(B) gel-filtration analysis of m1b3.(C) elisa assay of CH2 clone.(D) clone ring 2 and ring 3 sequence of m1b3, it mainly has the form of monomer compared with original CH2 sequence (SEQ ID NO:97-100).

Figure 17 shows the figure neutralized by the pseudovirus of the clone from the second CH2 library.Analytical separation is cloned in the neutralising capacity of concentration for the HIV pseudovirus of identical group of 100 μ g/ml from three of the second library.The ScFv X5 of the parallel purification of the concentration of 20 μ g/ml is with comparing.

Figure 18 is the figure of the CH2 binding substances of Identification display conserved epitope.The CH2 being mainly monomer clones m1b3 and is biotin labeling and it is used in competitive ELISA measures.ELISA antigen Bal gp120-CD4 is coated on the bottom of elisa plate.The biotinylated m1b3 of 1.7 of fixed amount μMs is mixed with unlabelled m1b3, scFv X5 or m36-Fc of indicatrix and adds each hole to.The m1b3 combined is detected with streptavidin-HRP.

Sequence table

The nucleotide sequence listed in subsidiary sequence table and aminoacid sequence be use as in 37C.F.R.1.822 the standard letter abbreviation of nucleotide base that defines and amino acid whose three letter code illustrate.Only show a chain of each nucleotide sequence, but complementary strand be interpreted as by the chain of any display mentioned and be included.In the sequence table of enclosing:

SEQ ID NO:1 is mankind V _hthe aminoacid sequence of structural domain.

SEQ ID NO:2-4 is the V of three-type-person's antibody-like _hthe aminoacid sequence of structural domain.

SEQ ID NO:5 is the aminoacid sequence of mankind γ 1CH2 structural domain.

SEQ ID NO:6-10 is the nucleotide sequence of the PCR primer in library for generation of sudden change CH2 structural domain.

SEQ ID NO:11-30 is the aminoacid sequence of the fragment of the sudden change CH2 structural domain with random ring 1.

SEQ ID NO:31-50 is the aminoacid sequence of the fragment of the sudden change CH2 structural domain with random ring 3.

SEQ ID NO:51-68 is the nucleotide sequence for the CDR3 from people's antibody being moved into the PCR primer in CH2 support.

SEQ ID NO:69-87 is the aminoacid sequence of the fragment of the through engineering approaches CH2 structural domain of the H3 with transplanting.

SEQ ID NO:88 and 89 is aminoacid sequences of the fragment of CH2 structure domain mutant m01.

SEQ ID NO:90 and 91 is aminoacid sequences of the fragment of CH2 structure domain mutant m02.

SEQ ID NO:92 is the aminoacid sequence of mouse CH2.

SEQ ID NO:93 is the aminoacid sequence of CH2 ring 1.

SEQ ID NO:94 is the consensus amino acid sequences of mutant CH2 ring 1.

SEQ ID NO:95 is the aminoacid sequence of CH2 ring 3.

SEQ ID NO:96 is the consensus amino acid sequences of mutant CH2 ring 3.

SEQ ID NO:97 is the aminoacid sequence of the CH2 ring 2 from clone m1a1.

SEQ ID NO:98 is the consensus amino acid sequences of the mutant CH2 ring 2 from clone m1a1.

SEQ ID NO:99 is the aminoacid sequence of the CH2 ring 3 from clone m1a1.

SEQ ID NO:100 is the consensus amino acid sequences of the mutant CH2 ring 3 from clone m1a1.

SEQ ID NO:101-105 is the nucleotide sequence of the PCR primer for a CH2 library of increasing.

SEQ ID NO:106 is the aminoacid sequence of m1a1 synthetic peptide.

SEQ ID NO:107 is the aminoacid sequence of m1a1 ring 1.

SEQ ID NO:108 is the aminoacid sequence of m1a2 ring 1.

Embodiment

I. abridge

ADCC: the cytotoxicity of antibody dependent cellular mediation

CDC: CDC

CDR: complementary determining region

DNA: thymus nucleic acid

ELISA: enzyme-linked immunosorbent assay

HIV: human immunodeficiency virus

Ig: immunoglobulin (Ig)

NK: natural killer cell

RNA: Yeast Nucleic Acid

SDR: specificity determining residue

II. term

Except as otherwise noted, otherwise technical term uses according to common usage.In molecular biology, the definition of generic term is found in Genes V (gene V) (the ISBN 0-19-854287-9) of the Benjamin Lewin published for 1994 by Oxford University Press; People's (volume) The Encyclopedia of Molecular Biology (molecular biology encyclopedia) (ISBN 0-632-02182-9) such as the Kendrew published in 1994 by Blackwell Science Ltd; And by VCH Publishers, Inc. at the Robert A.Meyers (volume) that nineteen ninety-five publishes, Molecular Biology and Biotechnology:a Comprehensive Desk Reference (molecular biology and biotechnology: comprehensive desk reference) (ISBN 1-56081-569-8).

In order to promote describing of each embodiment of the present invention, provide the following explanation to concrete term:

Use: by selected approach, composition is incorporated in experimenter.Such as, if selected approach is intravenously, so use composition by the vein that composition is incorporated into experimenter.

Animal: lived many cells vertebrate organism, comprises the classification of such as Mammals and bird.Term mammal comprises people and non-human animal.Similarly, term " experimenter " comprises people and veterinary subject.

Antibody: comprise substantially by the albumen (or albumen composition) of one or more polypeptide of the fragment coding of immunoglobulin gene or immunoglobulin gene.The immunoglobulin gene identified comprises κ, λ, α, γ, δ, ε and μ constant region gene and countless immune globulin variable region genes.Light chain is classified as κ or λ.Heavy chain is classified as γ, μ, α, δ or ε, they so that respectively define immunoglobulin class: IgG, IgM, IgA, IgD and IgE.

Basic immunoglobulin (Ig) (antibody) structural unit is generally the tetramer.Each tetramer comprises two to identical polypeptide chain, and often pair has " gently " chain (about 25kDa) and " weight " chain (about 50-70kDa).The N end of every bar chain defines about 100 to 110 of primary responsibility antigen recognition or with last amino acid whose variable region.Term " variable light chain (V _l) " and " variable heavy chain (V _h) " refer to these light chains and heavy chain respectively.Every bar light chain contains single constant domain (CL), and every bar heavy chain contains three constant domain CH1, CH2 and CH3 (or for IgE and IgM four constant domain).See the schematic diagram of the conventional immune globulins molecule of Figure 1A.

As used in this, term " antibody " comprises complete immunoglobulin (Ig) and some have the fragment of the well-characterized of about 25 to 100kD molecular weight.Such as, Fab, Fv of combining with target protein (or with the epi-position in albumen or fusion rotein) and scFv (scFv) will be also the specific-binding agents of this albumen (or epi-position).These antibody fragments are defined as foloows: (1) Fab, the fragment of the monovalent antigen binding fragment containing antibody molecule, and it produces by obtaining a part for complete light chain and a heavy chain with the whole antibody of papain digestion; (2) Fab ', by with the whole antibody of pepsin, reduction subsequently obtains the part of complete light chain and heavy chain and the fragment of antibody molecule that obtains; Each antibody molecule obtains two Fab ' fragments; (3) (Fab ') ₂, by with the whole antibody of pepsin and the fragment of the antibody not having reduction subsequently to obtain; (4) F (ab ') ₂, the dimer of two the Fab ' fragments connected together by two disulfide linkage; (5) Fv, the genetically engineered fragment being expressed as two chains of the variable region containing light chain and the variable region of heavy chain; And (6) scFv, single-chain antibody, the genetically engineered chemoattractant molecule of the variable region containing light chain, the variable region of heavy chain, the variable region of described light chain is connected by the applicable peptide linker as gene fusion single chain molecule with the variable region of heavy chain.The method preparing these fragments is conventional (see such as, Harlow and Lane, Using Antibodies:A Laboratory Manual (using antibody: laboratory manual), CSHL, New York, 1999).

Antibody can be mono-clonal or polyclonal.By means of only citing, monoclonal antibody can be prepared from murine hybridoma according to the classical way of Kohler and Milstein (Nature 256:495-97,1975) or its deriving method.Such as, the detailed step produced for monoclonal antibody is described (Using Antibodies:A Laboratory Manual (using antibody: laboratory manual), CSHL, New York, 1999) by Harlow and Lane.

" humanization " immunoglobulin (Ig) such as humanized antibody is the immunoglobulin (Ig) comprising people's framework region and one or more CDR from non-human (such as mouse, rat or synthesis) immunoglobulin (Ig).Non-human immunoglobulin's called after " donor " of CDR is provided, and the human normal immunoglobulin called after " acceptor " of framework is provided.In one embodiment, all CDR are from the donor immunoglobulin in Humanized immunoglobulin." humanized antibody " is the antibody comprising humanization light chain and humanized heavy chain immuno's sphaeroprotein, such as Humanized monoclonal antibodies.Humanized antibody is bonded to and provides on the same or analogous antigen of the donor antibody of CDR.The acceptor framework of Humanized immunoglobulin can have the aminoacid replacement taking from donor framework limited the number.Humanized molecule can have extra conserved amino acid and replace, and described conserved amino acid replaces not to be affected in fact antigen combination or other immunoglobulin (Ig) functions.These molecules can build (for example, see United States Patent (USP) 5,585,089) by genetically engineered means.

Antigen: can the compound of the generation of antibody or t cell response in stimulating animal, composition or material, comprises the composition being injected or absorbing in animal.The product of antigen and specific humoral immunity or specific cellular immunity reacts.

Autoimmune disease: the immunity system wherein risen because of tissue injury produces the disease of the immunne response (such as, B cell response or t cell response) for the antigen (namely autoantigen) of the part for normal host.Autoantigen can available from host cell, or can available from the commensal normally surely growing mucomembranous surface, such as microorganism (being called commensal).

Affect mammiferous exemplary autoimmune disease and comprise rheumatoid arthritis, the few joint type sacroiliitis (juvenile oligoarthritis) of juvenile form, Collagen-induced Arthritis, Adjuvant Induced Arthritis, sjogren syndrome, multiple sclerosis, experimental autoimmune encephalomyelitis, inflammatory bowel (such as Crohn's disease, ulcerative colitis), autoimmunity gastratrophy, pemphigus vulgaris, psoriasis, vitiligo, type 1 diabetes, non-obese patients with type Ⅰ DM, myasthenia gravis, Grave is sick, struma lymphomatosa, sclerosing cholangitis, hardening sialadenitis, systemic lupus erythematous, autoimmune thrombocytopenic purpura, Goodpasture syndrome (Goodpasture ' s syndrome), Addison is sick, Systemic sclerosis, polymyositis, dermatomyositis, autoimmune hemolytic anemia, pernicious anemia and similar disease.

Binding affinity: the power of the combination of (such as, antibody, CH2 structural domain or CH3 structural domain and antigen or epi-position between) between binding site and part.Binding site X to the avidity of part Y by dissociation constant (K _d) represent, dissociation constant is the concentration of the Y needed for binding site occupying the half X existed in solution.Lower (K _d) show that avidity stronger or higher between x and y interacts, and need the part of low concentration to occupy these sites.In general, binding affinity by one or more amino acid whose change, modification in the epi-position identified by paratope (identifying the part of the molecule of epi-position) and/or can replace impact.Binding affinity can be the avidity of antibodies bind antigen.

In an example, binding affinity is measured by the endpoint titration in being measured by Ag-ELISA.If modify/end point titres difference at least 4 times compared with unaltered epi-position of specific antibody of the epi-position of displacement, as at least 10 times, at least 100 times or larger, so binding affinity is that one or more amino acid whose modification in epi-position by being identified by complementary antibody position and/or replace reduces (maybe can reduce with measuring) substantially.

CH2 or CH3 structural domain molecule: available from the polypeptide (or nucleic acid of coded polypeptide) of immunoglobulin (Ig) CH2 or CH2 structural domain.Immunoglobulin (Ig) can be IgG, IgA, IgD, IgE or IgM.In an embodiment as herein described, CH2 or CH3 structural domain molecule comprises at least one CDR or its function fragment.CH2 or CH3 structural domain molecule can also comprise extra aminoacid sequence, such as complete Gao Bianhuan.In another embodiment, CH2 or CH3 structural domain molecule at least have with CDR or its function fragment displacement one or more ring regions at least partially.In embodiments more as herein described, CH2 or CH3 structural domain comprises one or more sudden change in the ring region of this molecule." the ring region " of CH2 or CH3 structural domain refers to the protein part (such as, each CH2 structural domain comprises seven beta sheet A to G, and direction holds C to hold from N) between the region of beta sheet.As shown in Fig. 3 A-3C, CH2 structural domain comprises six ring regions: ring 1, ring 2, ring 3, ring A-B, ring C-D and ring E-F.Ring A-B, ring C-D and ring E-F lay respectively between beta sheet A and B, between C and D and between E and F.Ring 1,2 and 3 lays respectively between beta sheet B and C, between D and E and between F and G.For the Amino Acid Range of the ring in CH2 structural domain see table 1.CH2 and CH3 structural domain molecule disclosed herein can also comprise N end disappearance, and such as about 1 to about 7 amino acid whose disappearances.In concrete example, N holds disappearance to be 1,2,3,4,5,6 or 7 amino acid length.CH2 and CH3 structural domain molecule disclosed herein can also comprise C end disappearance, and such as about 1 to about 4 amino acid whose disappearances.In concrete example, C holds disappearance to be 1,2,3 or 4 amino acid length.

The size of CH2 and CH3 structural domain molecule is less, is usually less than 15kD.The size of CH2 and CH3 structural domain molecule can change, and this depends on that the CDR/ height inserted in ring region becomes the length of aminoacid sequence, the quantity inserting CDR and another molecule (as effector molecule or mark) and whether is combined with CH2 or CH3 structural domain.In some embodiments, CH2 and CH3 structural domain molecule does not comprise extra constant domain (i.e. CH1 structural domain or another CH2 structural domain or CH3 structural domain) or variable domains.In one embodiment, CH2 structural domain is from IgG, IgA or IgD.In another embodiment, constant domain is the CH3 structural domain from IgE or IgM with the CH2 domain homology of IgG, IgA or IgD.

CH2 and CH3 structural domain molecule provided herein can be glycosylation or nonglycosylated.Such as, recombinant C H2 structural domain or CH3 structural domain can be expressed in suitable mammalian cell to allow the glycosylation of this molecule.

Complementary determining region (CDR): the short amino acid sequence found in the variable domains of antigen receptor (as immunoglobulin (Ig) and φt cell receptor) albumen, it provides the contact site of antigen and the specificity to specific antigen thereof for this receptor.Each polypeptide chain of antigen receptor contains three CDR (CDR1, CDR2 and CDR3).Antigen receptor is normally made up of two polypeptide chains (heavy chain and light chain), therefore each antigen receptor have six can with the CDR of antigen contact.Because the most of sequence variations relevant to antigen receptor are found in CDR, so these regions are sometimes referred to as high structure changes territory.

CDR in the ring region of antigen receptor (usually between the region of beta sheet structure; See Fig. 3 A-3C) be found.These ring regions are commonly referred to Gao Bianhuan.Each antigen receptor comprises six Gao Bianhuan: H1, H2, H3, L1, L2 and L3.Such as, H1 ring comprises the CDR1 of heavy chain, and L3 ring comprises the CDR3 of light chain.CH2 and CH3 structural domain molecule as herein described comprises the amino acid of the immigration from antibody variable territory.The amino acid moved into comprises CDR at least partially.The amino acid moved into can also comprise extra aminoacid sequence, as complete Gao Bianhuan.As used herein, " function fragment " of CDR be the CDR of the ability remaining binding specificity antigen at least partially.

The coding rule of CDR position is by people such as Kabat., (1991) Sequences of Proteins of Immunological Interest (sequence of immunology target protein), 5th edition, NASA, public health service, NIH, Bethesda, MD (NIH publication number 91-3242) describe.

Contact: with direct physical association place, it comprise in solid form and in liquid form the two.

Degeneracy variant: as described herein, " the degeneracy variant " of CH2 or CH3 structural domain molecule is the polynucleotide of coding CH2 or CH3 structural domain molecule, and these polynucleotide comprise the sequence due to genetic code degeneration.There are 20 kinds of natural amino acids, they are most of is specified by more than one codon.Therefore, all degenerate core nucleotide sequences are included, as long as the aminoacid sequence of CH2 or the CH3 structural domain molecule coded by this nucleotide sequence is constant.

Structural domain: a kind of protein structure, this structure remains the tertiary structure of the rest part independent of protein.In some cases, the separative functional performance of structural domain tool and can be added, remove or be transferred to another albumen and not have loss function.

Effector molecule: expect, to the cell of molecule or chimeric molecule institute target, there is the molecule of the effect of expectation or the part of chimeric molecule.Effector molecule is also referred to as effect part (EM), therapeutical agent or diagnostic reagent or similar terms.

Therapeutical agent comprises this compounds, as nucleic acid, albumen, peptide, amino acid or derivative, glycoprotein, radio isotope, lipid, carbohydrate or recombinant virus.Exonuclease treatment part and diagnosis of partial comprise antisense nucleic acid, with the derivative oligonucleotide of strand or double-stranded DNA covalent cross-linking and the oligonucleotide forming triple helix.Alternatively, the molecule be connected with targeting moiety such as CH2 or CH3 structural domain molecule can be encapsulated system containing therapeutic composition, as liposome or micella, described therapeutic composition such as medicine, nucleic acid (as antisense nucleic acid) or conductively-closed can avoid the other treatment part being directly exposed to the recycle system.The method that preparation is connected to the liposome of antibody well known to a person skilled in the art.See such as, United States Patent (USP) the 4th, 957, No. 735; And the people such as Connor., Pharm.Ther.28:341-365,1985.Diagnostic reagent or diagnosis of partial comprise radio isotope or other detectable marks.Detectable mark for this type of object is also well known in the art, and comprise radio isotope (as ³²p, ¹²⁵i and ¹³¹i), fluorophore, chemistry are given out light agent and enzyme.

Epi-position: antigenic determinant.Having the specified chemical group on antigenic molecule (namely this molecule causes specific immune response), or continuous or discrete peptide sequence.The specific epitope of antibodies is the epi-position of three-dimensional structure based on this antibody and coupling (or homology).

Express: translated nucleic acid is protein.Protein can be expressed and stay in cell, becomes the component of cell surface membrane, or is secreted in extracellular matrix or medium.

Expression control sequenc: the nucleotide sequence of the expression of regulation and control heterologous nucleic acid sequence, described heterologous nucleic acid sequence is operably connected with it.When expression control sequenc control and regulate and control nucleotide sequence transcribe and (time suitable) translate time, this expression control sequenc is operably connected with this nucleotide sequence.Therefore expression control sequenc can comprise suitable promotor, enhanser, transcription terminator, initiator codon (i.e. ATG) before the gene of proteins encoded, the splicing signal of intron, the maintenance sequence allowing the correct open reading-frame (ORF) of this gene of the suitable translation of mRNA and terminator codon.Term " control sequence " is intended to minimumly comprise the component that its existence can affect expression, and comprises it and there is favourable additional component, such as leader sequence and fusion partner sequence.Expression control sequenc can comprise promotor.

Promotor instructs a series of nucleic acid control sequence of transcribed nucleic acid.Promotor comprises the necessary nucleotide sequence of near transcriptional start sites, as the TATA element in polymerase Il type promoter situation.Promotor also optionally comprises Distal enhancer or repressor element, and they can be positioned at distance transcription initiation site and reach many kilobases to place.Comprise both constitutive promoter and inducible promoter (see such as, the people such as Bitter, Methods in Enzymology (Enzymology method) 153:516-544,1987).

Also comprise those promoter elements being enough to give promotor dependent gene and expressing, it is that cell type specificity, tissue specificity are controlled that described promotor dependent gene is expressed, or by external signal or reagent derivable; This class component can be arranged in 5' or the 3' region of gene.Comprise both constitutive promoter and inducible promoter (see such as, the people such as Bitter, Methods in Enzymology (Enzymology method) 153:516-544,1987).Such as, when cloning in bacterial system, inducible promoter can be used, as pL, plac, ptrp, ptac (ptrp-lac hybrid promoter) and the similar promotor of bacteriophage λ.In one embodiment, when cloning in mammalian cell system, the genomic promotor (as metallothionein promoter) available from mammalian cell or the promotor from mammalian virus can be used (as retrovirus long terminal repeat; Adenoviral late promotor; Vaccinia virus 7.5K promotor).The promotor produced by recombinant DNA or synthetic technology also can be used to provide transcribing of nucleotide sequence.

Polynucleotide can be inserted in the expression vector containing promoter sequence, described promoter sequence promotes effectively transcribing of the insertion gene order of host.Expression vector is usually containing replication orgin, promotor and the specific nucleic acid sequence of permission to the Phenotypic Selection of transformant.

Framework region: the aminoacid sequence inserted between CDR (or hypervariable region).Framework region comprises variable light chain framework region and variable heavy chain framework regions.Each variable domains comprises four framework regions, is commonly referred to R1, FR2, FR3 and FR4.Framework region is used for CDR being remained on suitable direction and combines for antigen.Framework region forms beta sheet structure usually.

Fungi related antigen (FAA): the fungal antigen of the immunne response that fungi specific T-cells can be stimulated to limit.Exemplary FAA includes but not limited to from following antigen: Candida albicans (Candida albicans), genera cryptococcus (Cryptococcus) are (as from d25 or MP98 of Cryptococcus neoformans (C.neoformans) or MP88, or its immune fragment), Blastomyces (Blastomyces) (such as, as Blastomyces dermatitidis (B.dermatitidis), WI-1 or its immune fragment) and Histoplasma (Histoplasma) (as Histoplasma capsulatum (H.capsulatum)).

Allos: the polypeptide of allos or polynucleotide refer to polypeptide available from different sources or species or polynucleotide.

Hypervariable region: the extra high region of the sequence variability in antibody variable territory.Hypervariable region forms the ring structure between the beta sheet of framework region.Therefore, hypervariable region is also referred to as " Gao Bianhuan ".Each variable domains comprises three hypervariable regions, is commonly referred to H1, H2 and H3, and is commonly referred to L1, L2 and L3 in heavy chain in light chain.The ring structure of Gao Bianhuan is depicted in Fig. 3 A-5C.

Immunne response: immune cell is if B cell, T cell, scavenger cell or polymorphonuclear cell (polymorphonucleocyte) are to the reaction of the stimulator of such as antigen.Immunne response can comprise any cell of the health participating in host defense, such as, secrete the epithelial cell of Interferon, rabbit or cytokine.Immunne response includes but not limited to innate immune responses or inflammation.

Immune conjugate: effector molecule and antibody or covalently bound with CH2 or CH3 structural domain molecule.Effector molecule can be detectable mark or immunotoxin.Concrete, the limiting examples of toxin include but not limited to: toxalbumin, ricin, Pseudomonas exotoxin (PE, as PE35, PE37, PE38 and PE40), diphtheria toxin (DT), the toxin of Toxins, botulin or its modification, or cell growth inhibiting or kill other toxic agent of cell directly or indirectly.Such as, PE and DT causes dead high toxicity compound by liver toxicity usually.But, PE and DT modification become the form as immunotoxin by such as under type: remove the natural target component (the B chain as structural domain Ia and DT of PE) of toxin and replace this natural target component with different targeting moieties (as CH2 or CH3 structural domain molecule).In one embodiment, CH2 or CH3 structural domain molecule is engaged with effector molecule (EM).In another embodiment, CH2 or the CH3 structural domain molecule engaged with effector molecule is also connected to improve CH2 or the CH3 structural domain molecule transformation period in vivo with other molecules of lipid or albumen or peptide.This connection can pass through chemical means or recombinant means." chemical means " refers to the reaction between CH2 or CH3 structural domain molecule and effector molecule, to make to form covalent linkage between these two molecules thus to form a molecule.Peptide linker (short peptide sequence) optionally can be comprised between CH2 or CH3 structural domain molecule and effector molecule.Because immune conjugate is by two molecules with different functionality at first, as antibody and the preparation of effector molecule, so they are sometimes referred to as " chimeric molecule ".Term used herein " chimeric molecule " therefore refers to the targeting moiety of combination (coupling) to effector molecule, as part, antibody or CH2 or CH3 structural domain molecule.

Term " combination ", " joint ", " bonding " or " connection " instigate two polypeptide to become a continuous peptide molecule, or refer to radioactive nucleotides or other molecules and polypeptide, as CH2 or CH3 structural domain molecule covalent connects.In concrete context, described term comprises assignment body, as antibody moiety engages with effector molecule (" EM ").

Immunogen: can the immunne response of stimulating animal under proper condition, as the compound of the generation of antibody or T cell immunne response, composition or material, comprises the composition being injected or absorbing in animal.

Be separated: " separation " biological components (as nucleic acid molecule or albumen) from the naturally occurring other biological component of this component (such as, the other biological component of cell) in isolated or purified is out substantially, described other biological component such as other chromosomal and extrachromosomal DNA and RNA and albumen, comprise other antibody.Comprised nucleic acid by the purification process purifying of standard and albumen by " separation " nucleic acid and albumen." antibody of separation " be its from other albumen or biological components substantially isolated or purified out to make to keep the antibody of its antigen-specific.This term also comprises by the nucleic acid of the recombinant expressed preparation in host cell and the nucleic acid of albumen (comprising CH2 and CH3 structural domain molecule) and chemosynthesis or albumen or its fragment.

Mark: directly or indirectly with other molecule as antibody or CH2 or CH3 structural domain molecule are combined the detectable compound or the composition that promote the detection of this molecule.Concrete, the limiting examples of mark comprise fluorescence labels, enzyme connects and radio isotope.

Ligand contact residue or specificity determining residue (SDR): the participation contact part in CDR or the residue of antigen.Ligand contact residue is also referred to as specificity determining residue (SDR).Non-ligand contact residue is the residue not contacting part in CDR.Non-ligand contact residue also can be Framework residues.

Nano antibody (nAb): by through engineering approaches to make CH2 or the CH3 structural domain molecule of molecular specificity conjugated antigen.Known smallest molecule based on antigen-specific binding antibody structural domain by through engineering approaches with CH2 and the CH3 structural domain molecule of conjugated antigen.

Tumorigenesis and tumour: neoplastic product is knurl (tumour), it divides by cell transition the tissue abnormalities produced to grow.Knurl is also referred to as " cancer ".The tumour do not shifted is also referred to as " optimum ".Invasion surrounding tissue and/or the tumour that can shift are also referred to as " pernicious ".

Noumenal tumour, the example as sarcoma and cancer comprises fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, and other sarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdosarcoma, colorectal carcinoma, lymphoid malignancies, carcinoma of the pancreas, mammary cancer, lung cancer, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, rodent cancer, gland cancer, syringocarcinoma, sebaceous carcinoma, papillary carcinoma, papillary carcinoma, medullary carcinoma, bronchiogenic cancer, renal cell carcinoma, liver cancer, cholangiocarcinoma, choriocarcinoma, Wei Ermusishi tumour, cervical cancer, carcinoma of testis, bladder cancer and central nerve neuroma are (as neurospongioma, astrocytoma, medulloblastoma, craniopharyngioma (craniopharyogioma), ependymoma, pinealoma, hemangioblastoma, acoustic tumor, oligodendroglioma, meningioma (menangioma), melanoma, neuroblastoma and retinoblastoma).

The example of neoplastic hematologic disorder comprises leukemia, and leukemia comprises acute leukemia (as acute lymphoblastic leukemia, acute myeloblastic leukemia, acute myelogenous leukemia and myeloblastosis, promyelocytic leukemia, myelomonocytic leukemias, monocarpotic cellularity and erythroleukemia); Chronic leukemia (as chronic myeloid (granulocytic) leukemia, chronic lymphocytic leukemia and lymphocytic leukemia); Polycythemia vera; Lymphoma; Hodgkin's disease; Non-Hodgkin lymphoma (painless form and advanced form); Multiple myeloma; Wal Dan Sitelunshi macroglobulinemia; Heavy chain disease; Myelodysplastic syndrome; Hairy cell leukemia; And osteomyelodysplasia.

Nucleic acid: the polymkeric substance that the analogue existed by the non-natural of the nucleotide units connected by phosphodiester bond (analogue that the non-natural of ribonucleotide, deoxyribonucleotide, relevant naturally occurring structural variant and synthesis thereof exists), relevant naturally occurring structural variant and synthesis thereof is formed.Therefore, this term comprises following nucleotide polymer: its nucleotide and the connection between them comprise the synthetic analogues that non-natural exists, such as such as and be not restricted to: thiophosphatephosphorothioate, phosphoramidate, methylphosphonate, chiral-methyl phosphonates, 2 '-O-methyl ribonucleotides, peptide-nucleic acid (PNA) and analogue.These type of polynucleotide can such as use automatization DNA synthesizer to synthesize.Term " oligonucleotide " typically refers to the short polynucleotide being no more than about 50 Nucleotide.Should understand when nucleotide sequence is represented by DNA sequence dna (i.e. A, T, G, C), this also comprises RNA sequence (i.e. A, U, G, C), " U " replacement " T " in RNA sequence.

Ordinary symbol is used to carry out described nucleotide sequence herein: the left hand end of single stranded nucleotide sequence is 5' end; The left-hand of Double-stranded nucleotide sequence is to being called 5' direction.Nucleotide is added the direction of nascent RNA transcript to also referred to as transcriptional orientation with 5' to 3'.The DNA chain with the sequence identical with mRNA is called " coding strand "; On the DNA chain of the identical sequence of the mRNA had with transcribe from this DNA and the sequence that 5' to the 5' being positioned in rna transcription thing holds be called " upstream sequence "; On the DNA chain with the sequence identical with mRNA and be encoding RNA transcript 3' to 3' end sequence be called " downstream sequence ".

" cDNA " refers to the strand complementary or identical with mRNA or the DNA of double chain form.

" coding " refers at polynucleotide, the natural characteristics that particular sequence as the Nucleotide in gene, cDNA or mRNA serves as other polymkeric substance in synthesising biological process or macromolecular template and the biological nature therefrom produced, other polymkeric substance described or macromole have the nucleotide sequence (i.e. rRNA, tRNA and mRNA) determined or the aminoacid sequence determined.Therefore, if transcribing and translating of the mRNA produced by gene creates albumen in cell or other biological system, so this albumen of this genes encoding.Albumen or other products of encode this gene or cDNA is can be described as both the coding strand (its nucleotide sequence is identical with mRNA sequence and be usually provided in sequence table) of the transcription templates of gene or cDNA and noncoding strand.Unless otherwise, otherwise " nucleotide sequence of encoding amino acid sequence " comprise for merger form each other and all nucleotide sequences of identical aminoacid sequence of encoding.The nucleotide sequence of proteins encoded and RNA can comprise intron.

" recombinant nucleic acid " refers to the nucleic acid with following nucleotide sequence: described nucleotide sequence non-natural are bonded together and the sequence section be originally separated by artificial combination two and preparing.Artificial combination is normally by chemosynthesis, or the nucleic acid fragment (such as passing through genetic engineering technique) be more usually separated by manual operation is realized.Recombinant nucleic acid comprises nucleic acid carrier, nucleic acid that is that described nucleic acid carrier comprises the amplification that can be used to transform the host cell be applicable to or that assemble.The host cell comprising recombinant nucleic acid is called " recombinant host cell ".Then by this genetic expression in recombinant host cell to produce " recombinant polypeptide ".Recombinant nucleic acid also can play non-coding function (such as, promotor, replication orgin, ribosome bind site and analogue).

Be operably connected: when the first nucleotide sequence is placed together with the second nucleotide sequence with functional relationship, this first nucleotide sequence and this second nucleotide sequence are operatively connected.Such as, if promotor affects transcribing of encoding sequence or expresses, so this promotor is operably connected with this encoding sequence.In general, the DNA sequence dna be operably connected is continuous print, and if be necessary joint two protein-coding regions, then the DNA sequence dna be operably connected is in identical reading frame.

Pathogenic agent: cause the biology of the disease of host or illness former.Pathogenic agent comprises such as bacterium, virus, fungi, protozoon and parasite.Pathogenic agent is also referred to as infectious agent.

The example of Causative virus comprises the virus in following Viraceae: Retroviridae (Retroviridae) (such as human immunodeficiency virus (HIV); Human T-cell's property leukosis virus (HTLV); Picornaviridae (Picornaviridae) (such as poliovirus, hepatitis A virus, hepatitis C virus, enterovirus, human coxsackievirus, rhinovirus, Chinese mugwort can virus, hoof-and-mouth disease is viral); Caliciviridae (Calciviridae) (such as causing the strain of gastro-enteritis); Togaviridae (Togaviridae) (such as equine encephalitis, rubella virus); Flaviviridae (Flaviridae) (such as dengue virus, flavivirus, west nile virus, Saint Louis' encephalitis virus, encephalitis b virus and other encephalitiss); Coronaviridae (Coronaviridae) (such as coronavirus, serious acute respiratory Distress syndrome (SARS) virus; Rhabdoviridae (Rhabdoviridae) (such as stomatitis herpesvirus, rabies virus); Filoviridae (Filoviridae) (such as Ebola virus); Paramyxoviridae (Paramyxoviridae) (such as parainfluenza virus, mumps virus, Measles virus, respiratory syncytial virus (RSV)); Orthomyxoviridae family (Orthomyxoviridae) (such as influenza virus); Bunyaviridae (Bunyaviridae) (such as Hantaan virus, Xin Nuowa virus, Rift valley fever virus, bunyavirus, Phlebovirus and Nairovirus); Arenaviridae (Arena viridae) (hemorrhagic fever virus, Machupo virus, turtledove be virus rather); Reoviridae (Reoviridae) (such as reovirus, Orbivirus and rotavirus); Birnavirus section (Birnaviridae); Hepadnaviridae (Hepadnaviridae) (hepatitis B virus); Parvoviridae (Parvoviridae) (parvovirus); Papovaviridae (Papovaviridae) (papilloma virus, polyomavirus, BK virus); Adenoviridae (Adenoviridae) (most of adenovirus); Herpetoviridae (Herpesviridae) (hsv (HSV)-1 and HSV-2; Cytomegalovirus (CMV); Epstein-Barr virus (EBV); Varicella zoster virus (VZV); And other simplexviruss, comprise HSV-6); Poxviridae (Poxviridae) (variola virus, vaccinia virus, poxvirus); With Iridoviridae (Iridoviridae) (as African swine fever virus); Filoviridae (Filoviridae) (such as Ebola virus, Marburg virus); Caliciviridae (Caliciviridae) (such as norwalk virus) and unfiled virus (cause of disease of such as spongiform encephalopathy, the cause of disease (being considered to hepatitis B defective type satellite virus) of δ hepatitis; And Astrovirus).

The example of fungal pathogens includes but not limited to: Cryptococcus neoformans (Cryptococcus neoformans), Histoplasma capsulatum (Histoplasma capsulatum), posadasis spheriforme (Coccidioides immitis), Blastomyces dermatitidis (Blastomyces dermatitidis), chlamydia trachomatis (Chlamydia trachomatis), Candida albicans (Candida albicans).

The example of bacterial pathogens includes but not limited to: helicobacter pylori (Helicobacter pyloris), Borrelia burgdoyferi (Borelia burgdorferi), legionella pneumophilia (Legionella pneumophilia), mycobacterium (Mycobacteria sps) is (as mycobacterium tuberculosis (M.tuberculosis), avian tuberculosis mycobacterium (M.avium), Mycobacterium intracellulare (M.intracellulare), mycobacterium kansasii (M.kansaii), mycobacterium gordonae (M.gordonae)), streptococcus aureus (Staphylococcus aureus), Diplococcus gonorrhoeae (Neisseria gonorrhoeae), Neisseria meningitidis (Neisseria meningitidis), Listeria monocytogenes (Listeria monocytogenes), streptococcus pyogenes (Streptococcus pyogenes) (A group streptococcus), streptococcus agalactiae (Streptococcus agalactiae) (B group streptococcus), suis (Streptococcus) (grass green population), streptococcus faecium (Streptococcus faecalis), streptococcus bovis (Streptococcus bovis), suis (anaerobism kind), streptococcus pneumoniae (Streptococcus pneumoniae), pathogenic Campylobacter bacterium (pathogenic Campylobacter sp.), enterococcus bacteria (Enterococcus sp.), Haemophilus influenzae (Haemophilus influenzae), Bacillus anthracis (Bacillus anthracis), corynebacterium diphtheriae (corynebacterium diphtheriae), coryneform bacteria (corynebacterium sp.), erysipelothrix rhusiopathiae (Erysipelothrix rhusiopathiae), clostridium perfringens (Clostridium perfringers), clostridium tetani (Clostridium tetani), enteroaerogen (Enterobacter aerogenes), klepsiella pneumoniae (Enterobacter aerogenes), pasteurella multocida (Pasturella multocida), bacterioide (Bacteroides sp.), Fusobacterium nucleatum (Fusobacterium nucleatum), Streptobacillus moniliformis (Streptobacillus moniliformis), Treponoma palladium (Treponema pallidium), superfine treponema (Treponema pertenue), leptospira (Leptospira) and actinomyces Israeli (Actinomyces israelli).

Other pathogenic agent (as protobiont) comprising: plasmodium falciparum (Plasmodium falciparum) and toxoplasma gondii (Toxoplasma gondii).

Pharmaceutically acceptable medium: pharmaceutically acceptable carrier (medium) useful is in this disclosure conventional.Remington ' s Pharmaceutical Sciences (Lei Mingdun pharmaceutical science), E.W.Martin, Mack publishing company, Easton, PA, the 15th edition (1975) describe composition and the preparation of the drug delivery being suitable for one or more therapeutic compounds or molecule (as one or more antibody) and other medicaments.

In general, the kind of carrier will depend on the concrete method of application of employing.Such as, parenteral administration comprises injectable liquids usually, described injectable liquids comprise as medium pharmaceutically with acceptable fluid on physiology, as water, physiological saline, balanced salt solution, aqueous dextrose, glycerine etc.For solids composition (such as, powder, pill, tablet or capsule form), conventional non-toxic solid carrier can comprise such as: the N.F,USP MANNITOL of pharmaceutical grade, lactose, starch or Magnesium Stearate.Except biologically neutral carrier, pharmaceutical composition to be administered can contain a small amount of non-toxic auxiliary substances, as wetting agent or emulsifying agent, sanitas and pH buffer reagent etc., and such as sodium acetate or sorbitan monolaurate.

Polypeptide: a kind of polymkeric substance, wherein monomer is the amino-acid residue be bonded together by amido linkage.When amino acid is alpha amino acid, L-type optical isomer or D type optical isomer can be used." polypeptide " or " protein " is intended to contain any aminoacid sequence and the sequence comprising modification as the term is employed herein, as glycoprotein.Term " polypeptide " is specifically intended to the protein covering naturally occurring protein and restructuring or synthesis generation.

Term " residue " or " amino-acid residue " comprise the amino acid be incorporated in albumen, polypeptide or peptide of indication.

" guarding " aminoacid replacement is substantially do not affect or reduce the activity of polypeptide or those replacements antigenic.Such as, polypeptide can comprise about 1 at the most, about 2 at the most, at the most about 5, at the most about 10 or at the most about 15 conservative to replace, and the antibody that its specific binding is combined with former polypeptide.The variation that term is guarded also comprises the unsubstituted parent amino acid of amino-acid substitution using and replace, and condition is that the antibody (antibodies raised antibodies raised) that the antibody produced for the polypeptide replaced produces also reacts with unsubstituted polypeptide immune.The example that conservative property replaces shows below.

Conservative property replaces the general structure keeping (a) to replace polypeptide backbone in region, such as folding or helical conformation; B () is in the electric charge of the molecule of target spot or hydrophobicity; And/or the size of (c) side chain (bulk).

Be generally expected that the replacement of maximum change producing protein properties will be nonconservative, such as following change: (a) hydrophilic residue such as seryl or Threonyl replace (or being substituted by) hydrophobic residue such as leucyl, isoleucyl, phenylalanyl, valyl or alanyl; B () halfcystine or proline(Pro) replace (or being substituted by) any other residue; C residue such as lysyl, arginyl or histidyl-that () has electropositive side chain replace (or being substituted by) electronegativity residue such as glutamyl or aspartyl; Or the residue such as phenylalanine (d) with bulky side chain replaces the residue such as glycine that (or being substituted by) does not have side chain.

Prevent, treat or improve disease: " prevention " disease refers to the development completely suppressing disease." treatment " refers to the Results improving the S or S of disease or pathological state after disease starts to develop." improvement " refers to quantity or the severity of the S or S reducing disease.

Probe and primer: probe comprises the nucleic acid be separated being connected to detectable mark or reporter molecule.Primer is short nucleic acid and can is length to be 15 Nucleotide or longer DNA oligonucleotide.Primer can be annealed to complementary target dna strand to form crossbred between this primer and target dna strand by nucleic acid hybridization, and then it is extended along target dna strand by DNA polymkeric substance enzyme.Primer pair may be used for amplifying nucleic acid sequence, such as, by polymerase chain reaction (PCR) or other nucleic acid amplification methods known in the art.It will be understood by those skilled in the art that the specificity of concrete probe or primer increases with the increase of its length.Therefore, such as, the primer comprising 20 conserved nucleotides is annealed to target by with the specificity higher than the corresponding primer of only 15 Nucleotide.Therefore, in order to obtain larger specificity, can select to comprise probe and the primer of 20,25,30,35,40,50 an or more continuous nucleotide.

Purifying: term purifying do not need absolute purity; Otherwise it is intended to as a relative term.Therefore, such as, CH2 or the CH3 structural domain molecule of purifying is the molecule be separated from natural associated protein and other pollutents in whole or in part, wherein this molecule is relative to its natural existence, such as, relative to its purity in cell extract or biofluid, suitable degree can be purified to.

Term " purifying " comprises the product of expectation, as analogue or stand-in or other biological active compound, other compounds or part is wherein made to combine to allow the connection of other compounds with CH2 or CH3 structural domain molecule and/or be provided for the preparation of therapeutic treatment or diagnostor.

In general, CH2 or the CH3 structural domain molecule of purifying is included in before respective compound in the intact drug preparation that being used for the treatment of property uses mixes with other compositions or prepare substantially, all macromolecular species existed in goods more than 80%.Other compositions can comprise pharmaceutical carrier, vehicle, buffer reagent, absorption enhancer, stablizer, sanitas, adjuvant or other similar common compositions (co-ingredient).More typically, all macromolecular species becoming representative to exist in Purified preparations before mixing with other formulation ingredients CH2 or CH3 structural domain molecular purifications to be greater than 90%, to be usually greater than 95%.In other cases, the goods of purifying can be homogeneous in fact, and wherein other macromolecular species are less than 1%.

Recombinant chou: recombinant nucleic acid or polypeptide have the sequence of non-natural existence or have nucleic acid or the polypeptide of the sequence prepared by the sequence section of artificial combination two kinds separation originally.Artificial combination is normally realized by chemosynthesis or the nucleic acid segment (such as passing through genetic engineering technique) that is more usually separated by manual operation.

Sample: represent overall part, fragment or section.Any material contained in this term, comprises such as from the sample that experimenter obtains.

" biological sample " is the sample obtained from experimenter, includes but not limited to cell, tissue and body fluid.Body fluid comprises such as: the derivative of saliva, phlegm, celiolymph, urine, blood and blood and fraction, comprise serum and lymphocyte (as B cell, T cell and segmentation fraction thereof).Tissue comprises the tissue removed from the tissue of examination of living tissue thing, ptomatopsia thing and pathology sample and bioptic tissue or surgery, comprise such as loose, freezing, be fixed on tissue that is in formalin and/or that imbed in paraffin.

In particular embodiments, biological sample is as obtained blood or serum from experimenter.Biological sample normally obtains from Mammals, and described Mammals is as rat, mouse, ox, dog, cavy, rabbit or primates.In one embodiment, primates is macaque, chimpanzee or people.

Support: as used herein, CH2 or CH3 structural domain support can be used as introducing sudden change (such as in ring region; See Fig. 2 and Fig. 3 A-3C) platform so that the recombinant C H2 making antigen be combined with this CH2 or CH3 structural domain or CH3 structural domain.In some embodiments, support is changed the stability showing raising compared with natural CH2 or CH3 structural domain.In concrete example, cysteine residues pair introduced by mutagenesis support, thus allows the formation of one or more non-native disulfide bond.In some cases, support is CH2 or the CH3 structural domain with N end disappearance (according to appointment 1 to about 7 amino acid whose disappearance).

Sequence iden: the similarity between nucleotide sequence or aminoacid sequence is expressed according to the similarity between these sequences, is called sequence iden in addition.Sequence iden is normally measured according to percentage identities (or similarity or homology); This per-cent is higher, and these two sequences are more similar.Homologue or variant, when using standard method comparison, will have the sequence iden of relative high degree.

Sequence alignment method for comparing is known in the art.Various program and alignment algorithm are described in: Smith and Waterman, Adv.Appl.Math.2:482, and 1981; Needleman and Wunsch, J.Mol.Biol.48:443,1970; Pearson and Lipman, Proc.Natl.Acad.Sci.U.S.A.85:2444,1988; Higgins and Sharp, Gene73:237-244,1988; Higgins and Sharp, CABIOS 5:151-153,1989; The people such as Corpet., Nucleic Acids Research 16:10881-10890,1988; And the people such as Pearson and Lipman, Proc.Natl.Acad.Sci.U.S.A.85:2444,1988.Altschul., Nature Genet.6:119-129,1994.

Basic Local Alignment Search Tool (the BLAST of NCBI ^tM) (people such as Altschul., J.Mol.Biol.215:403-410,1990) can obtain from several source, comprise American National Biotechnology Information center (NCBI, Bethesda, MD) with for the internet be connected with sequence analysis programs blastp, blastn, blastx, tblastn and tblastx.

Specific-binding agent: the agent be substantially only combined with the target determined.Therefore, antigen specific binders is the agent be substantially combined with antigenic polypeptide or its anti-genic fragment.In one embodiment, specific-binding agent is the monoclonal antibody of specific binding antigenic polypeptide or its anti-genic fragment or polyclonal antibody or CH2 or CH3 structural domain molecule.

Relate to antigen, term " specific binding " refers to antibody or other ligand entity or is partly preferentially combined with the cell or tissue with this antigen and is not preferentially combined with the cell or tissue of this antigen lacking detectable amount.Certainly, the non-specific interaction recognizing to a certain degree can occur between molecule and non-target cell or non-target tissue.However, specific binding can be divided into by mediating the specific recognition of antigen.Specific binding causes the combination between antibody (or CH2 or CH3 structural domain molecule) and the cell with this antigen to be greatly better than combination between the antibody (or CH2 or CH3 structural domain molecule) of combination and the cell lacking this antigen.Specific binding usually cause the antibody that combines or CH2 or CH3 structural domain molecule (time per unit) to the amount of cell or tissue with this antigenic polypeptide compared to the antibody combined or CH2 or CH3 structural domain molecule (time per unit) to the amount of cell or tissue correspondingly lacking antigenic polypeptide, improve and be greater than 2 times, as being greater than 5 times, be greater than 10 times or be greater than 100 times.Binding proteins specific matter needs antibody according to selecting the specificity of concrete albumen or CH2 or CH3 structural domain molecule under such conditions.Panimmunity measures antibody or CH2 or the CH3 structural domain molecule that form is suitable for selecting to carry out specific immune response with concrete albumen.Such as, solid phase ELISA immunoassay are used routinely.

Experimenter: lived multicellular organisms, comprises vertebrate organism, this classification comprises people and non-human mammal.

Treatment significant quantity: be enough to the amount reaching this agent of desired effects in the experimenter treated with specific dose.This type of agent comprises CH2 or CH3 structural domain molecule as herein described.Such as, this can be the amount of HIV specific C H2 structural domain molecule for preventing, treating or improve HIV.Ideally, the treatment significant quantity of antibody is the amount being enough to prevent, treat or improve the infection of experimenter or the disease such as caused by HIV and not causing great cytotoxic effect.For prevent, improve and/or treat experimenter agent treatment significant quantity by depend on be treated experimenter, the type of sufferer and the method for application of seriousness and therapeutic composition.

Toxin: to the cytotoxic molecule of cell.Toxin includes but not limited to: the toxin of toxalbumin, ricin, Pseudomonas exotoxin (PE), diphtheria toxin (DT), Toxins, botulin, saporin, restrictocin or gelonin or its modification.Such as, PE and DT causes dead high toxicity compound by liver toxicity usually.But, PE and DT modification can be become the form as immunotoxin, this is natural target component (the B chain of structural domain Ia and DT of such as PE) by removing toxin and replaces this natural target component with different targeting moieties (as CH2 or CH3 structural domain molecule).

Transduction: the cell of transduction nucleic acid molecule has been incorporated into cell wherein by Protocols in Molecular Biology.As used herein, all technology nucleic acid molecule can introduced in this kind of cell are contained in term transduction, comprise with virus vector transfection, transform and accelerate to introduce naked DNA by electroporation, fat transfection and particle gun with plasmid vector.

Tumor associated antigen (TAA): the tumour antigen of the immunne response that tumor specific T cells can be stimulated to limit.Example T AA includes but not limited to RAGE-1, tyrosine oxidase, MAGE-1, MAGE-2, NY-ESO-1, Melan-A/MART-1, glycoprotein (gp) 75, gp100, beta-catenin, PRAME, MUM-1, WT-1, CEA and PR-1.Other TAA be known in the art (for example, see people such as Novellino., Cancer Immunol.Immunother.54 (3): 187-207,2005) and comprise the TAA also do not identified.

Carrier: introduce in host cell, produce the nucleic acid molecule of the host cell of conversion thus.Carrier can comprise its nucleotide sequence copied in host cell of permission, as replication orgin.Carrier can also comprise one or more selectable marker gene and other Genetic elements known in the art.

VAA (VAA): the virus antigen of the immunne response that virus specific t cell can be stimulated to limit.Exemplary VAA includes but not limited to the antigen from following virus: human immunodeficiency virus (HIV), BK virus, JC virus, Epstein-Barr virus (EBV), cytomegalovirus (CMV), adenovirus, respiratory syncytial virus (RSV), hsv 6 (HSV-6), parainfluenza virus 3 or Type B influenza.

Unless explained in addition, otherwise all technical terms used herein and scientific terminology have the implication identical with the implication that those skilled in the art understand usually.Unless context clearly indicates in addition, otherwise singular references comprises plural referents.Similarly, unless context clearly indicates in addition, otherwise word "or" be intended to comprise " with ".So " comprising A or B " represents comprise A or B, or A and B.Will also be understood that for all base sizes given by nucleic acid or polypeptide or amino acid size and all molecular weight or molecular mass values be all approximation, and provide for description.Although method that is similar with material with method as herein described or that be equal to and material can be used in enforcement of the present invention or test, described below applicable method and material.All publications herein, patent application, patent and other reference are incorporated to herein with its entirety all by reference.In the case of a conflict, comprise the explanation of term, will be as the criterion with this specification sheets.In addition, described material, method and example are only exemplary, and and not intended to be limiting.

III. the general introduction of several embodiment

Conventional antibody is the large-scale multimeric protein mixture comprising at least four polypeptide chains, and described four polypeptide chains comprise two light chains and two heavy chains (schematic diagram see the conventional immune globulins of Figure 1A).The heavy chain of antibody and light chain contain the variable region of conjugated antigen and provide the constant region (as CH1, CH2 and CH3 structural domain) of support structure and effector function.Antigen binding domain comprises two structural domains be separated, heavy-chain variable domains (V _h) and light variable domains (V _l).Typical antibody such as IgG molecule has the molecular weight of about 150kD.After protease digestion, identified the many less Fab (such as, Fab, Fab' and F (ab ') of naturally occurring antibody ₂).These antibody fragments have the molecular weight of scope at about 50kD to 100kD.Used recombination method to produce optional Fab, be called single chain variable fragment (scFv), it is the V connected by the peptide linker by synthesis _land V _hcomposition.ScFv molecule has the molecular weight of about 25-30kD.

But in some cases, the therapeutic of antibody or antibody fragment uses may be limited due to the size of antibody.Such as, if antibody or antibody fragment are too large, then tissue infiltration and epi-position enter and may be restricted.In addition, many therapeutic antibodies are inhuman sources, and this can produce toxicity in human experimenter.In view of these limitation, can the small-sized human antibodies of specific binding antigen be utilize desired by the diagnostic use of antibody or its fragment or treatment use.

This document describes engineered antibody constant domain molecules.Disclosed herein is the recombinant C H2 as support and CH3 structural domain molecule, described support is for introducing sudden change to make antigen be combined with this molecule.Additionally provide CH2 and the CH3 structural domain molecule of the modification of specific binding antigen.In some embodiments, antibody constant structural domain is the CH2 structural domain from IgG, IgA or IgD.In other embodiments, antibody constant structural domain is the CH3 structural domain from IgE or IgM.Disclosed CH2 and CH3 structural domain molecule is little, stable, solvable, has minimum toxicity and even does not have toxicity, and in some cases can conjugated antigen.CH2 and CH3 structural domain molecule as herein described does not comprise more than one constant domain and does not comprise immunoglobulin variable domain territory.

There is provided herein the polypeptide comprising immunoglobulin (Ig) CH2 or CH3 structural domain, wherein said CH2 or CH3 structural domain comprises at least one complementary determining region from heterologous immunoglobulin variable domains (CDR) or its function fragment (as SDR).Additionally provide CH2 or the CH3 structural domain molecule comprising at least one sudden change (as 1,2,3,4,5,6,7,8,9,10 or more sudden changes) in one or more rings of CH2 or CH3 structural domain.CH2 or CH3 structural domain molecule as herein described has the molecular weight being less than about 15kD.In some embodiments, CH2 or CH3 structural domain molecule has the molecular weight of about 12kD to about 14kD.In some embodiments, CH2 or CH3 structural domain comprises about 1 to about 7 amino acid, cuts as 1,2,3,4,5,6 or 7 amino acid whose N holds level with both hands.In some embodiments, CH2 or CH3 structural domain molecule comprises about 1 to about 4 amino acid, cuts as 1,2,3 or 4 amino acid whose C holds level with both hands.

Specific sudden change is introduced CH2 or CH3 structural domain and/or allos CDR to move into CH2 or CH3 structural domain and make this polypeptide can conjugated antigen.In some embodiments, the immigration part from heterologous immunoglobulin only comprises CDR or its function fragment.In other embodiments, immigration part comprises extra sequence, as all or part of of Gao Bianhuan.The length moving into part can be different, but normally between 5 to 21 amino acid, comprise 5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20 or 21 amino acid.In one embodiment, immigration part is between 8 to 15 amino acid.Although the length moving into part is different, CH2 or the CH3 structural domain molecular specificity conjugated antigen obtained.In some embodiments, CH2 or CH3 structural domain molecule is with about 10 ^-6, about 10 ^-7or about 10 ^-8the K of M _dspecific binding antigen.In some embodiments, polypeptide comprises more than one CDR or its function fragment, as two or three CDR.

In some embodiments, being replaced by CDR or its function fragment at least partially of ring region of CH2 or CH3 structural domain.The amino acid whose number removed from ring region can be different.In some embodiments, the amino acid whose number removed from ring region is between 1 to 10 amino acid, comprises 1,2,3,4,5,6,7,8,9 or 10 amino acid.In other embodiments, move into CDR and do not remove amino acid from ring region.The amino acid whose number removed from one or more CH2 or CH3 structural domain ring can be different.Those skilled in the art can pass through experience, such as, determined the suitable sequence that will remove by the ability of the stability of test CH2 or CH3 structural domain molecule, solvability and binding purposes antigen.

The concrete CDR moved into can be from any immunoglobulin variable domain territory, as V _hstructural domain or V _lany CDR of structural domain.In one embodiment, CDR is CDR1.In another embodiment, CDR is CDR2.In another embodiment, CDR is CDR3.In another embodiment, two or three or more CDR be moved in the ring of CH2 or CH3 structural domain molecule.

By CDR CH2 or the CH3 structural domain ring (or CDR move into wherein and do not remove the CH2 structural domain of ring sequence) of replacing can be any ring of this CH2 or CH3 structural domain.In some embodiments, ring region is selected from ring 1, ring 2, ring 3, ring A-B, ring C-D or ring E-F.Any ring of CH2 or CH3 structural domain can be replaced by any CDR.In addition, multiple ring can be replaced with arbitrary combination by CDR.In one embodiment, ring 1 is replaced by CDR1 or CDR3.In another embodiment, ring 3 is replaced by CDR1 or CDR3.In another embodiment, ring 1 and ring 3 are replaced by CDR1 and CDR3 respectively.In another embodiment, ring 1 and ring 3 are replaced by CDR3 and CDR1 respectively.In another embodiment, ring A-B is replaced by CDR1; Ring C-D is replaced by CDR2; Or ring E-F is replaced by CDR3.

In preferred embodiments, polypeptide provided herein does not comprise variable domains, as V _hstructural domain or V _lstructural domain.

Antibody constant structural domain can available from the immunoglobulin (Ig) of any type.In one embodiment, immunoglobulin (Ig) is IgG.In other embodiments, immunoglobulin (Ig) is IgA, IgD, IgM or IgE.In specific examples, constant domain is the CH2 structural domain from IgG.

In some embodiments, CH2 or the CH3 structural domain of conjugated antigen has the extra sudden change improving this stability of molecule.Such as, molecule can comprise and allows to form the sudden change of non-native disulfide bond, the form of described non-native disulfide bond be such as by introducing a pair aminoacid replacement to replace Original Residue with cysteine residues.In some instances, hold in A chain at the N of constant domain and introduce the first aminoacid replacement, and introduce the second aminoacid replacement in the C end G chain of constant domain.In addition, the antigen in conjunction with CH2 and CH3 structural domain molecule can be glycosylated or nonglycosylated.

Additionally provide the polypeptide comprising the immunoglobulin (Ig) CH2 structural domain of IgG, Ig or IgD or the CH3 structural domain of IgE and IgM herein, wherein said CH2 structural domain or CH3 structural domain comprise the first aminoacid replacement and the second aminoacid replacement, wherein said first aminoacid replacement and each personal cysteine residues of the second aminoacid replacement replace original residue, wherein said cysteine residues forms disulfide linkage, and wherein said polypeptide has the molecular weight being less than about 15kD.This type of CH2 and CH3 structural domain shows the stability of the raising of CH2 and the CH3 structural domain relative to unmodified, and is therefore used as sudden change to introduce with the support making antigen be combined with CH2 or CH3 structural domain.

In some embodiments, the first aminoacid replacement holds in A chain at N, and the second aminoacid replacement holds in G chain at C, and this permission forms disulfide linkage (schematic diagram see Fig. 3 A-3C ring region) between A chain and G chain.In some instances, constant domain is the CH2 structural domain of IgG.

In specific examples as herein described, the first aminoacid replacement is L12 to C12, and the second aminoacid replacement is K104 to C104 (numbering about SEQ ID NO:5).In other instances, the first aminoacid replacement is V10 to C10, and the second aminoacid replacement is K104 to C104 (numbering about SEQ ID NO:5).

CH2 and CH3 structural domain support can comprise the extra sudden change such as improving stability or strengthen solubleness and expression.In some embodiments, CH2 or CH3 structural domain comprise about 1 to about 7 amino acid whose N hold level with both hands cut.In concrete example, N hold level with both hands cut be 1,2,3,4,5,6 or 7 amino acid length.In some embodiments, CH2 or CH3 structural domain support comprise about 1 to about 4 amino acid whose C hold level with both hands cut.In concrete example, C hold level with both hands cut be 1,2,3 or 4 amino acid.

In some embodiments, also mutagenesis CH2 or CH3 structural domain support combine to give antigen.In a particular embodiment, at least one ring of (i) CH2 or CH3 structural domain is suddenlyd change; (ii) being replaced by from the CDR of heterologous immunoglobulin variable domains or its fragment at least partially of ring region of CH2 or CH3 structural domain; Or (iii) the two has concurrently.

In addition, CH2 or CH3 structural domain can be nonglycosylated or glycosylated.Such as, recombinant C H2 or CH3 structural domain can be expressed to allow posttranslational modification in mammalian cell, as glycosylation.

In some embodiments, antigen from pathogenic agent, as virus or bacterium.In one embodiment, pathogenic agent is HIV.In other embodiments, antigen is cancer-specific antigen or cancer-associated proteins.In other embodiments, antigen is relevant to autoimmune disease, such as TNF-α.

In some embodiments, CH2 or CH3 structural domain molecule is in conjunction with tumour antigen.Tumour antigen can be tumor associated antigen well known in the art.

Additionally provide the composition comprising CH2 or CH3 structural domain molecule as herein described herein.In some embodiments, described composition comprises CH2 structural domain or CH3 structural domain and pharmaceutically acceptable carrier.

Additionally provide the nucleic acid molecule of disclosed CH2 or the CH3 structural domain molecule of coding herein, comprise the carrier of this nucleotide sequence and comprise the cell of this carrier.

In some embodiments, through engineering approaches CH2 or CH3 structural domain molecule comprise Fc receptor binding site and can in conjunction with at least one Fc acceptors.In specific examples, Fc acceptor is neonatal Fc receptor.Effector function is given to CH2 or CH3 structural domain molecule by the ability in conjunction with Fc acceptor, such as such as ADCC.In other embodiments, through engineering approaches CH2 or CH3 structural domain combine can the complement associated molecule of complement activation system, as C1q.In other embodiments other, CH2 or CH3 structural domain molecular juction is bonded to effector molecule, and described effector molecule includes but not limited to treatment part, diagnosis of partial or test section.

Additionally provide the method using CH2 or CH3 structural domain molecule to prepare medicine.In one embodiment, medicine is used for the treatment of HIV.In another embodiment, medicine is used for the treatment of cancer.In another embodiment, medicine is used for the treatment of autoimmunization illness or inflammatory condition.

CH2 or CH3 structural domain molecule as herein described can by through engineering approaches with the antigen of any expectation of specific binding.The method of qualification and selection antigen-specific CH2 or CH3 structural domain molecule can use any applicable technology known in the art, such as, by using phage display library to realize.

There is provided herein and a kind ofly identify the recombinant C H2 structural domain of specific binding target antigen or the method for CH3 structural domain.Described method comprises: (a) is provided in the library of their displaying on surface recombinant C H2 or the particle of CH3 structural domain, and wherein said CH2 or CH3 structural domain has the molecular weight being less than about 15kD; B the library of described particle is contacted the particle selecting target antigen described in specific binding by () with target antigen; And (c) clones CH2 or CH3 structural domain nucleic acid molecule from the particle of expression specificity in conjunction with CH2 or the CH3 structural domain of described target antigen, identify CH2 or the CH3 structural domain of target antigen described in specific binding thus.In some embodiments, library produces as follows: (i) provides the library of the nucleic acid molecule of the genetic diversity colony of coding CH2 or CH3 structural domain, and wherein said genetic diversity colony provides by sudden change being incorporated in one or more ring regions of described CH2 or CH3 structural domain; And (ii) library of express nucleic acid molecule in recombinant host cell, thus described CH2 structural domain or CH3 structural domain to be expressed on the surface of described particle and to be encoded described CH2 or CH3 structural domain nucleic acid molecule by the genetic stocks of described particle.In some embodiments, CH2 or CH3 structural domain comprises about 1 to about 7 amino acid whose N end disappearance.In some embodiments, particle is bacteriophage particles.

In some embodiments, phage library expresses recombinant C H2 structural domain, such as IgGCH2 structural domain.In some embodiments, CH2 structural domain or CH3 structural domain be included in ring 1 at least one sudden change or in ring 2 at least one sudden change in ring 3 at least one sudden change or in ring A-b at least one sudden change in ring C-D at least one sudden change or in ring E-F at least one sudden change or its arbitrary combination.

Any applicable recombinant host cell can be used to produce bacteriophage particles.This type of host cell is well known in the art.In some instances, recombinant host cell is TG1 cell.

Additionally provide a kind of method preparing the library of recombinant C H2 or CH3 structural domain herein, described method comprises (i) and sudden change is introduced in one or more ring regions of CH2 structural domain or CH3 structural domain support, or (ii) by a part of replacing the ring region of described CH2 structural domain or CH3 structural domain support from the CDR of heterologous immunoglobulin variable domains or its function fragment, or (iii) the two has concurrently, wherein said support comprises the immunoglobulin (Ig) CH2 structural domain of the separation of IgG, IgA or IgD, or the CH3 structural domain of IgE or IgM.

In some embodiments, CH2 or CH3 structural domain support also comprises about 1 to about 7 amino acid, and such as about 1,2,3,4,5,6 or 7 amino acid whose N holds level with both hands and cuts.In some embodiments, CH2 or CH3 structural domain support also comprises about 1 to about 4 amino acid, and such as about 1,2,3 or 4 amino acid whose C holds level with both hands and cuts.

In some cases, CH2 or CH3 structural domain support also comprises the extra sudden change of stable molecule.In some embodiments of the method, described CH2 or CH3 structural domain support also comprises the first aminoacid replacement and the second aminoacid replacement, the each personal cysteine residues of wherein said first and second aminoacid replacement replaces original residue, and wherein said cysteine residues forms disulfide linkage.

Additionally provide and a kind ofly identify the recombinant C H2 structural domain of specific binding target antigen or the method for CH3 structural domain, described method is comprised the library making to be produced by method disclosed herein and contacts recombinant C H2 or CH3 structural domain to select target antigen described in specific binding with described target antigen.

Additionally provide the library of CH2 or CH3 structural domain molecule, such as phage display library.Library comprises and has one or more sudden change, CDR, Gao Bianhuan of immigration or CH2 or the CH3 structural domain molecule of its function fragment.The library comprising Mutated residues can be used to identify CH2 or the CH3 structural domain molecule of the antigen-binding affinity with expectation and/or identify immunogenic CH2 or the CH3 structural domain molecule with minimizing.

Additionally provide the test kit comprising CH2 or CH3 structural domain molecule disclosed herein.In one embodiment, CH2 or CH3 structural domain molecule is labeled (such as using fluorescent mark, radio-labeling or enzyme labelling).In another embodiment, test kit comprises the illustrative material of the using method of open CH2 or CH3 structural domain molecule.Illustrative material may be written, (the such as computer format floppy or CD) of electronic form or may be visual (such as video file).Test kit can also comprise the additional component of the special applications promoted test kit design.Therefore, such as test kit can extraly containing certification mark means (such as enzyme labelling enzyme substrates, detect fluorescently-labeled filter set, suitable second mark as second antibody, etc.).Test kit can comprise the conventional buffer reagent for implementing concrete grammar and other reagent extraly.This type of test kit and suitable content are that those skilled in the art is known.

IV. engineered antibody constant domain

Engineered antibody constant domain molecules as herein described small-sized (being usually less than 15kD), this is detection, Diagnosis and Treat provides significant advantage.Such as, the small size of molecule allows larger epi-position to enter and better tissue infiltration.As shown in figure 5 c, CH2 domain antibodies provided in this article has antibody lower than other types and antibody fragment, the such as molecular weight of scFv, Fab and IgG molecule.They are also less than V _hdomain antibodies.

As described herein, can to there is not under other immunoglobulin domains comprising variable domains or other constant domain conjugated antigen effectively in CH2 or CH3 structural domain molecule.Such as, CH2 or CH3 structural domain molecule can with about 10 ^-6, about 10 ^-7, about 10 ^-8, or about 10 ^-9or less kD specific binding antigen.

CH2 or the CH3 structural domain of specific binding antigen as herein described comprise from immunoglobulin variable domain territory at least one heterologous amino acid sequence and/or comprise at least one sudden change.The heterologous amino acid sequence moved in CH2 or CH3 structural domain comprises at least one CDR or its function fragment (such as from the SDR of the antibody of specific binding object antigen).The aminoacid sequence moved into containing end and/or the extra aminoacid sequence to the extension of C end from CDR to N, such as, can also comprise other amino acid of Gao Bianhuan.Therefore, in some embodiments, through engineering approaches CH2 or CH3 structural domain molecule comprise the complete Gao Bianhuan from heterologous immunoglobulin variable domains.Through engineering approaches CH2 and CH3 structural domain can also comprise second or the 3rd CDR or Gao Bianhuan.The length of CDR or Gao Bianhuan moved into can be different.Suitable length can be determined by experience, such as, by expressing through engineering approaches CH2 or CH3 structural domain and evaluating stability and the solubility of albumen, and by determining binding affinity.Determine that albumen solubility is well known in the art with the method for the protein expression of assessment antigen-binding affinity.As described herein, determine that the sequence up to 21 amino acid lengths can successfully move into CH2 structural domain.

People CH2 structural domain comprises six ring regions: ring 1, ring 2, ring 3, ring A-B, ring C-D and ring E-F.CDR and/or Gao Bianhuan from heterologous immunoglobulin variable domains can arbitrary combination move in one or more these rings any (for example, see Fig. 5 A-5C).

The aminoacid sequence of people γ 1CH2 structural domain is listed as SEQ ID NO:5.The amino-acid residue comprising each ring region is shown in in following table 1.Start amino acid position number with the numbering 1 of first of CH2 residue.

Table 1

The amino acid position of the ring of CH2 structural domain

Ring	Amino acid position (SEQ ID NO:5)
		Ring A-B	14-27
Ring 1	35-43
		Ring C-D	54-62
Ring 2	67-69
		Ring E-F	78-88
Ring 3	96-100

People V _hthe aminoacid sequence of structural domain to be shown in Figure 1B and to list as SEQ ID NO:1.The amino-acid residue comprising each CDR and Gao Bianhuan is shown in in following table 2.

Table 2

The amino acid position of Gao Bianhuan

CDR/ ring	Amino acid position (SEQ ID NO:1)
		H1/CDR1	27-36
H2/CDR2	50-68
		H3/CDR3	99-109

In an exemplary embodiment, with the V from people's antibody _hthe height of structural domain becomes nine amino acid of 10 amino-acid substitutions from the ring 1 of CH2 structural domain of ring H1/CDR1.In other exemplary, with the V from people's antibody _hthe height of structural domain becomes six amino acid of 12 or 13 amino-acid substitutions from the ring 3 of CH2 structural domain of ring H3/CDR3.In another exemplary embodiment, with the V from people's antibody _hthe height of structural domain becomes six amino acid of 10 amino-acid substitutions from the ring 3 of CH2 structural domain of ring H1/CDR1.In other exemplary, with the V from people's antibody _hthe height of structural domain becomes nine amino acid of 12 or 13 amino-acid substitutions from the ring 1 of CH2 structural domain of ring H3/CDR3.

In other embodiments, with 5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20 or 21 amino acid of one or more CDR or Gao Bianhuan from heterologous antibody with arbitrary combination D-loop 1,2,3,0,1,2,3,4,5,6,7,8,9 or 10 one or more amino acid in A-B, C-D or E-F.CDR or Gao Bianhuan can from V _lor V _hstructural domain (see Fig. 3 A-3C).

The one or more ring moved into or one or more CDR can from any object antibody.This antibody-like includes but not limited to pathogen specific antibody and cancer specific antibody.Pathogen specific antibody comprises the antibody of such as specific binding antigen, and described antigen is from such as virus, bacterium or fungi, protozoon or parasitic pathogenic agent.In an exemplary embodiment, antibodies specific is in conjunction with HIV-1.Cancer specific antibody comprises specific recognition and is expressed (such as on cell surface) by cancer cells but the antibody of the antigen of not expressed by other non-cancerous cells.Cancer specific antibody includes but not limited to identify following antibody: lung cancer, mammary cancer, prostate cancer, liver cancer, bladder cancer, thyroid carcinoma, kidney, carcinoma of the pancreas, colorectal carcinoma, skin carcinoma, melanoma, neuroblastoma, Ewing's sarcoma, leukemia or lymphadenomatous cell or tissue.

In some embodiments, through engineering approaches CH2 or CH3 structural domain molecule comprise and have known specific CDR/ hypervariable sequence.Or through engineering approaches CH2 structural domain molecule can comprise one or more random CDR peptide sequence.The mutation analysis that can carry out CDR identifies the immunogenic CH2 structural domain molecule of binding affinity and/or the reduction with raising.In addition, CH2 or the CH3 structural domain molecule combining specific purpose antigen as described below with high-affinity is identified in the library that can produce CH2 or the CH3 structural domain molecule comprising CDR peptide sequence that is random or sudden change.

Such as the object for the treatment of, diagnose or detect, CH2 and CH3 structural domain molecule provided in this article can also comprise effector molecule.Such as, effector molecule can comprise toxin and detectable mark, such as radio-labeling, enzyme or fluorescent marker.The following describe other details (see " effector function of antibody constant structural domain molecule ") of the effector molecule type that can use together with CH2 with CH3 structural domain molecule.

V. antibody constant structural domain molecular library

Additionally provide the through engineering approaches CH2 of cdr amino acid sequence that is that comprise radom insertion or that suddenly change or the library of CH3 structural domain molecule herein.Described library can be used to screen CH2 or CH3 structural domain molecule object specific antigen to high-affinity.In one embodiment, library is phage display library.Antibody phage display library and to produce the method in this type of library be (see such as, United States Patent (USP) the 6th, 195, No. 866, is incorporated to this paper by reference by 828, No. 422 and the 7th) well known in the art.

Describe the exploitation (United States Patent (USP) the 6th, 828, No. 422) in the library of polypeptide (comprising antibody).In order to produce the library (such as the library of CH2 or CH3 structural domain molecule) of polypeptide, the nucleotide sequence being suitable for creating library first must be produced.In order to produce this type of random nucleotide sequence, usually use fallibility PCR.Sudden change is introduced at random at least one ring.Such as, identify the set (such as two or three or more) of homologous protein, establish the database of protein sequence, and protein sequence is compared each other.When CH2 structural domain molecule, identify the set of people CH2 domain sequence and be used for creation database.Be used for this database being limited to the subgroup of the protein sequence showing high similarity in sequence and/or structural arrangement.For each subgroup, derive the peptide sequence comprising at least one consensus sequence, described consensus sequence represents the member of this subgroup (such as the subgroup of CH2 structural domain).The full set of peptide sequence represents the complete structural library of the set of homologous protein (CH2 structural domain).Can analyze with between the amino acid of qualification in described peptide sequence or disadvantageous interaction between described peptide sequence and other peptide sequences it according to the structural performance of these artificial polypeptide sequences.Therefore can by changing consensus sequence by this type of interaction removing.

Next, analyze peptide sequence with recognin element, comprise structural domain, ring, beta sheet, alpha-helix and/or CDR.Aminoacid sequence reverse translation is become corresponding nucleic acid sequence encoding, and the codon that this nucleic acid sequence encoding is suitable for being designed for the host expressing described nucleotide sequence uses.Set up one group of cleavage site with not two sites occurring of second time in each subsequence flanking nucleic acid sequences of the sub-element identified as previously discussed that makes to encode.This can realize in the following way: the cleavage site being tested and appraised side joint subsequence, or by changing one or more Nucleotide to produce cleavage site, and by removing this site from the remainder of gene.Cleavage site should be common to the sub-element of all correspondences or subsequence, and this allows the complete modularization arrangement producing the complete modularization arrangement of the subsequence in nucleotide sequence and the sub-element in corresponding polypeptide.

Above-mentioned nucleotide sequence uses any one synthesis of several method well known in the art, is such as such as synthesized by full genome or by the method for PCR-based.

In one embodiment, nucleotide sequence is cloned in carrier.Carrier can be sequencing vector well known in the art, expression vector or display carrier (such as phage display).Carrier can be included in a nucleotide sequence in similar and different operator or two or more nucleotide sequences.If in identical operon, nucleotide sequence separately can be cloned or is cloned as continuous sequence.

In one embodiment, one or more subsequences (such as ring) of nucleotide sequence are by different sequence substitutions.This can pass through to use as under type realizes: such as by suitable Restriction Enzyme, use adjacent with subsequence or excised by subsequence at the cleavage site of subsequence end, and by the different sequence substitutions subsequences compatible from the nucleotide sequence cut.In further embodiment, the different sequences (also referred to as " sequence of immigration ") of replacing original one or more subsequences are genome sequences of genome sequence from heterologous antibody or rearrangement, such as CDR, SDR or Gao Bianhuan.In some embodiments, heterologous sequence is stochastic sequence.Variability is incorporated in polypeptide (or CH2 structural domain molecule) to create library by the introducing of stochastic sequence.Any one that can use many methods well known in the art, to produce stochastic sequence, such as, by being used in the mixture using mononucleotide or trinucleotide in the few nucleic acid synthesis of automatization, or is undertaken by fallibility PCR.According to the amino acids distribution determining position in known protein sequence, stochastic sequence can be completely random, or is partial to some codon, or departs from some codon.In addition, the set of random subsequence can comprise the codon of different number, produces the set with the sub-element of different lengths.

Can under felicity condition known in the art from the carrier be applicable to express nucleic acid sequence.In one embodiment, the polypeptide of expressing from nucleotide sequence is screened.Can also be optimized polypeptide.Screening can be undertaken by using any method well known in the art, the Analytical system of polysome technology, enzymic activity or protein stability that the phage that such as phage display, selectivity infect, screening combine.Can by nucleotide sequence or amino acid sequence analysis or identified the polypeptide (such as CH2 structural domain molecule) with desired characteristic by mass spectrum.The desired characteristic of the polypeptide of screening can be such as to best avidity or the specificity of target molecule.

In some embodiments, phagemid vector can be used to express a large amount of nucleotide sequences, those nucleotide sequences in the library of CH2 or CH3 structural domain molecule of such as encoding (see such as, U.S. Patent Application Publication No. 2008/0312101) simultaneously.The known any screening assay method screening that can be applicable to phage can be used to express the library of the bacteriophage particles of CH2 and CH3 structural domain.Such as, phage can be exposed to solvable or fixing (such as onboard or on pearl) purifying antigen or be exposed to full cell, tissue or animal, so that qualification adheres to the phage of the target (such as, being combined with cancer cells or the antigen in virus particle) existed in composite structure and in special position relevant on physiology or in treatment.

Can will wherein clone, express and phagemid vector selected by specific isolation heterologous sequence extracts from bacterial cell to the combination of particular ligand based on it, and check order, pcr amplification and/or be again cloned into another suitable carrier, such as, for recombinant production large-scale in bacterium, plant, yeast or mammalian cell.

And the interactional detection of particular target antigen can be carried out by the Panning methods of application standard or by applying for assessment of the interactional more complicated Biophysical techniques between CH2 or the CH3 binding molecule shown and its target antigen, and described Biophysical techniques is such as based on spectrography or microscopy, phosphatase reaction or other high-throughput techniques of fluorescence.

After the expression CH2 of specific binding target antigen or the bacteriophage particles of CH3 structural domain are selected, can to be separated the phage of restructuring and relevant DNA sequence dna according to methods known in the art and characterize (such as, use Restriction Enzyme be separated from phagemid vector, direct Sequencing and/or pass through pcr amplification).Then these sequences can be transferred in the carrier be more suitable for, for modifying and/or express protokaryon or eukaryotic host cell further.The DNA sequence dna of coding CH2 or CH3 structural domain is once be inserted in applicable carrier to be namely incorporated in suitable host cell by any applicable means (conversion, transfection, joint, protoplast fusion, electroporation, calcium phosphate precipitation, direct microinjection etc.) and carry out transformant.

Then the set of this DNA molecular can be used to create the library of CH2 or CH3 structural domain molecule.Aforesaid method can be used to optimize the avidity of CH2 or CH3 structural domain molecule.Library can be used for identifying one or more CH2 or the CH3 structural domain molecules be combined with target.The qualification of CH2 or the CH3 structural domain molecule expected comprises expresses CH2 or CH3 structural domain molecule, and then screens them to be separated one or more molecules that the avidity expected is combined with given target molecule.If necessary, the modular design of DNA molecular allows the one or more hereditary subsequence of excision and replaces with the second subsequence of one or more coding structure sub-element.The step that then can repeat to express and screen is until produce CH2 or the CH3 structural domain molecule with the avidity of expectation.

Wherein use cleavage site to replace the method in one or more hereditary subunit (such as, one or more CH2 or CH3 structural domain ring region) by the random collection (library) of sequence in one embodiment.Then for the library that any selected antigen selection obtains.Select, collecting CH2 or the CH3 structural domain molecule with the characteristic (such as having the binding affinity of expectation) of expectation also can used as the parent material in next library.

In another embodiment, fusion rotein can be produced by providing the DNA sequence dna of both coding polypeptide as previously discussed and extra section.This type of part comprises immunotoxin, enzyme, effector molecule, treatment molecule, mark or label (such as detecting and/or purifying).

The polypeptide additionally providing nucleotide sequence, the carrier containing nucleotide sequence, the host cell containing carrier herein and produce according to aforesaid method.

Additionally provide one or more the test kit in nucleotide sequence, recombinant vectors, polypeptide and/or the carrier comprised according to aforesaid method, and for generation of polypeptide be applicable to host cell.

VI. the nucleic acid of encoding antibody constant domain molecule

The nucleotide sequence of coding CH2 or CH3 structural domain molecule and/or immunotoxin can by comprising the clone of such as suitably sequence or being prepared by any applicable method of direct chemosynthesis, described chemosynthesis by the following method, such as: the people such as Narang., Meth.Enzymol.68:90-99, the phosphotriester method of 1979; The people such as Brown., Meth.Enzymol.68:109-151, the approach of 1979; The people such as Beaucage, Tetra.Lett.22:1859-1862, the diethylphosphoryl amino acid method of 1981; By Beaucage & Caruthers, Tetra.Letts.22 (20): 1859-1862, the 1981 solid phase phosphoramidite triester methods described, the method uses as people such as such as Needham-VanDevanter, Nucl.Acids Res.12:6159-6168, the Automatic analyzer described in 1984; And the solid phase method of No. the 4th, 458,066, United States Patent (USP).Chemosynthesis creates single stranded oligonucleotide.This can by being converted into double-stranded DNA with complementary sequence hybridization or by use strand to do polyreaction that template archaeal dna polymerase carries out.Although technician will recognize that the chemosynthesis of DNA is generally restricted to the sequence of about 100 bases, longer sequence can obtain by connecting comparatively short data records.

The Exemplary core coding sequences that coding CH2 or CH3 structural domain molecule or coding comprise the immunotoxin of CH2 or CH3 structural domain molecule is prepared by clone technology.The example of suitable Cloning and sequencing technology and be enough to guidance technology personnel and illustrated see people, above Berger and Kimmel (volume) and above Ausubel such as above Sambrook by the instruction of many cloning practices.Product information from biological reagent manufacturers and experimental installation manufacturers also provides useful information.This type of manufacturers comprises SIGMA Chemical Company (Saint Louis, MO), R & D Systems (Minneapolis, MN), Pharmacia Amersham (Piscataway, NJ), CLONTECH Laboratories, Inc. (Palo Alto, CA), Chem Genes Corp., Aldrich Chemical Company (Milwaukee, WI), Glen Research, Inc., GIBCO BRL Life Technologies, Inc. (Gaithersburg, MD), Fluka Chemica-Biochemika Analytika (Fluka Chemie AG, Buchs, Switzerland), Invitrogen (San Diego, and Applied Biosystems (Foster City CA), and other commercial source many known to the skilled CA).

Nucleic acid can also be prepared by amplification method.Amplification method comprise polymerase chain reaction (PCR), ligase chain reaction (LCR) (LCR), based on the amplification system of transcribing (TAS), self-sustained sequence replication system (3SR).Kind widely cloning process, host cell and amplification in vitro method is that technician is known.

In an example, the CH2 structural domain molecule of use prepares by being inserted in the carrier of the cDNA comprising coded actions thing molecule (EM) by the cDNA of this CH2 structural domain molecule of coding.Carry out this insertion to make variable region and EM to be read in frame thus to make to produce a kind of polypeptide continuously.Therefore, the polypeptide of coding contains function CH2 domain region and function EM region.In an example, the cDNA of coded actions thing molecule (such as but not limited to cytotoxin) is connected to CH2 structural domain molecule with the carboxyl terminal making EM be positioned at this CH2 structural domain molecule.In an example, the cDNA (it is suddenlyd change to eliminate or reduces non-specific binding) of encoding Pseudomonas exotoxin (" PE ") is connected to CH2 structural domain molecule with the aminoterminal making EM be positioned at CH2 structural domain molecule.

After the separate nucleic acid of CH2 structural domain molecule (or immunotoxin) of encoding and clone, can by protein expression at recombined engineering cell, such as, in bacterium, plant, yeast, insect or mammalian cell.Such as, can by DNA being transferred in applicable host cell one or more DNA sequence dnas of expressing coding CH2 structural domain molecule in vivo.Cell can be protokaryon or eucaryon.This term also comprises the filial generation of any target host cell.Should understand all filial generations can not be identical with parental cell, because may deposit the sudden change occurred in a replication process.The method of stable transfer known in the art, remains in host continuously by foreign DNA.Or, the DNA sequence dna of encoding immune toxin, antibody or its fragment can be expressed in vitro.

The polynucleotide sequence of coding CH2 or CH3 structural domain molecule can be operably connected with expression control sequenc.The expression control sequenc that connection is operably connected with encoding sequence is to make to realize the expression of this encoding sequence under the condition compatible with this expression control sequenc.Expression control sequenc can include but not limited to suitable promotor, enhanser, transcription terminator, initiator codon (such as ATG) before the gene of proteins encoded, the splicing signal of intron, the maintenance allowing the proper reading frame of this gene of the suitable translation of mRNA and terminator codon.

Can be inserted in expression vector by the polynucleotide sequence of coding CH2 or CH3 structural domain molecule, described expression vector includes but not limited to be operated to allow the insertion of sequence or introducing and plasmid, virus or other carriers that can be expressed in prokaryotic organism or eukaryote.Host can comprise microorganism, yeast, insect and mammalian organism.In prokaryotic organism, express the method with the DNA sequence dna of eucaryon sequence or virus sequence is well known in the art.The have virus of biological function and the plasmid DNA vectors that can express in host and copy be known in the art.

Can be undertaken by routine techniques well known by persons skilled in the art with recombinant DNA transformed host cell.When host is protokaryon (such as the intestinal bacteria) that can absorb DNA, competent cell, can by gather in the crops after exponential phase of growth and pass through CaCl subsequently ₂the cell preparation that method uses program well known in the art to process.Or, can MgCl be used ₂or RbCl.Conversion can also be carried out if desired after the protoplasma forming host cell, or is undertaken by electroporation.

When host is eukaryote, insertion or the virus vector of this kind of conventional mechanical program of the method for this kind of transfection DNA of such as calcium phosphate precipitation, such as microinjection, electroporation, the plasmid be enclosed in liposome can be used.Eukaryotic cell can also with the second exogenous DNA molecule of the polynucleotide sequence of encoding immune toxin, antibody or its fragment and selectable phenotype of encoding (the sick thymidine kinase gene of such as herpes simplex) cotransformation.Other method be use the eukaryotic viral vectors of such as simian virus 40 (SV40) or bovine papilloma virus to come transient infections or transforming eukaryotic cells and expressing protein (see such as, Eukaryotic Viral Vectors (eukaryotic viral vectors), cold spring harbor laboratory, Gluzman compiles., 1982).Those skilled in the art easily can use expression system, such as in cell for generation of the plasmid of albumen and carrier, described cell comprises higher eukaryotic cell, such as COS, CHO, HeLa and myeloma cell line.

The conventional means that can be separated with immunity by comprising preparative scale chromatography carries out the abstraction and purification of recombinant expressed polypeptide (such as CH2 structural domain molecule).After being expressed, recombinant expressed polypeptide can according to the standard program purifying of this area, described standard program such as ammonium sulfate precipitation, affinity column, column chromatography and similar program are (generally see R.Scopes, Protein Purification (protein purification), Springer-Verlag, N.Y., 1982).Disclosed herein is the homogeneous substantially pure composition had at least about 90% to 95%, and for medicinal object, 98% to 99% or higher homogeneity can be used.If used in treatment, once be purified, partly or after reaching desired homogeneity, polypeptide should be substantially free of intracellular toxin.

Become the method for suitable activity form to be described for expressing protein and/or refolding from such as colibacillary bacterium and be known, and can be applicable to antibody disclosed herein.See people such as Buchner, Anal.Biochem.205:263-270,1992; Pluckthun, Biotechnology 9:545,1991; The people such as Huse, Science 246:1275,1989; And the people such as Ward, Nature 341:544,1989, be all incorporated to by reference herein.

Usually, the heterologous protein of function that has from intestinal bacteria or other bacteriums can be separated from inclusion body, and needs to use strong denaturant solubilising and refolding subsequently.In solubilization step process, as known in the art, reductive agent must be there is to be separated disulfide linkage.The Examples of buffers with reductive agent is: 0.1M Tris pH 8,6M guanidine, 2mM EDTA, 0.3MDTE (dithioerythritol).Can by realizing renaturation by the albumen of sex change and reduction dilution (such as 100 times) to refolding buffer reagent.Exemplary buffer reagent is 0.1M Tris, pH8.0,0.5M L-arginine, 8mM oxidized glutathione (GSSG) and 2mM EDTA.

Except the method for restructuring, the peptide symthesis of standard can also be used to build CH2 and CH3 structural domain molecule disclosed herein on the whole or partly.The solid phase synthesis that length is less than about 50 amino acid whose polypeptide can realize by the C terminal amino acid of this sequence being connected to insoluble supporter, subsequently the order remaining amino acid added in this sequence.Technology for solid phase synthesis is described in Barany & Merrifield, The Peptides:Analysis, Synthesis, Biology (peptide: analysis, synthetic biology). the 2nd volume: Special Methods in Peptide Synthesis (concrete grammar in peptide symthesis), part A. 3-284 page; The people such as Merrifield, J.Am.Chem.Soc.85:2149-2156,1963; And the people such as Stewart, Solid Phase Peptide Synthesis (Solid phase peptide synthesis), the second edition, Pierce Chem.Co., Rockford, Ill., 1984.Longer albumen can be synthesized compared with the aminoterminal of short-movie section and carboxyl terminal by condensation.The method (such as, by using coupling reagent N, N'-dicyclohexylcarbodiimide) forming peptide bond by activating C-terminal is well known in the art.

VII. antibody constant structural domain molecule is used to be used for diagnosis or treatment

CH2 and CH3 structural domain molecule have for diagnose and/or treat much antibody can disease or illness in the great potential of any one.Such as, CH2 or CH3 structural domain molecule may be used for any other disease or the illness that Therapeutic cancer, communicable disease (such as virus, bacterium, fungi or parasitic infection), autoimmune disease, inflammatory condition or antibody or its fragment can be used as therapeutical agent.

In some embodiments, communicable disease is caused by virus, such as from the virus of one of following section: Retroviridae (such as human immunodeficiency virus (HIV); Human T-cell's property leukosis virus (HTLV); Picornaviridae (such as poliovirus, hepatitis A virus, hepatitis C virus, enterovirus, human coxsackievirus, rhinovirus, Chinese mugwort can virus, hoof-and-mouth disease is viral); Caliciviridae (such as causing the strain of gastro-enteritis); Togaviridae (such as equine encephalitis, rubella virus); Flaviviridae (such as dengue virus, flavivirus, west nile virus, Saint Louis' encephalitis virus, encephalitis b virus and other encephalitiss); Coronaviridae (such as coronavirus, serious acute respiratory Distress syndrome (SARS) virus; Rhabdoviridae (such as stomatitis herpesvirus, rabies virus); Filoviridae (such as Ebola virus); Paramyxoviridae (such as parainfluenza virus, mumps virus, Measles virus, respiratory syncytial virus (RSV)); Orthomyxoviridae family (such as influenza virus); Bunyaviridae (such as Hantaan virus, Xin Nuowa virus, Rift valley fever virus, bunyavirus, Phlebovirus and Nairovirus); Arenaviridae (hemorrhagic fever virus, Machupo virus, turtledove be virus rather); Reoviridae (such as reovirus, Orbivirus and rotavirus); Birnavirus section; Hepadnaviridae (hepatitis B virus); Parvoviridae (parvovirus); Papovaviridae (papilloma virus, polyomavirus, BK virus); Adenoviridae (most of adenovirus); Herpetoviridae (hsv (HSV)-1 and HSV-2; Cytomegalovirus (CMV); Epstein-Barr virus (EBV); Varicella zoster virus (VZV); And other simplexviruss, comprise HSV-6); Poxviridae (variola virus, vaccinia virus, poxvirus); With Iridoviridae (as African swine fever virus); Filoviridae (such as Ebola virus, Marburg virus); Caliciviridae (such as norwalk virus) and unfiled virus (cause of disease of such as spongiform encephalopathy, the cause of disease (being considered to hepatitis B defective type satellite virus) of δ hepatitis; And Astrovirus).

In other embodiments, communicable disease is caused by the bacterium of a type, and described bacterium is such as: helicobacter pylori, Borrelia burgdoyferi, legionella pneumophilia, mycobacterium is (as mycobacterium tuberculosis, avian tuberculosis mycobacterium, Mycobacterium intracellulare, mycobacterium kansasii, mycobacterium gordonae), streptococcus aureus, Diplococcus gonorrhoeae, Neisseria meningitidis, Listeria monocytogenes, streptococcus pyogenes (A group streptococcus), streptococcus agalactiae (B group streptococcus), suis (grass green population), streptococcus faecium, streptococcus bovis, suis (anaerobism kind), streptococcus pneumoniae, pathogenic Campylobacter bacterium, enterococcus bacteria, Haemophilus influenzae, Bacillus anthracis, corynebacterium diphtheriae, coryneform bacteria, erysipelothrix rhusiopathiae, clostridium perfringens, clostridium tetani, enteroaerogen, klepsiella pneumoniae, pasteurella multocida, bacterioide, Fusobacterium nucleatum, Streptobacillus moniliformis, Treponoma palladium, superfine treponema, leptospira and actinomyces Israeli.

In other embodiments, communicable disease is by fungus-caused, such as: Cryptococcus neoformans, Histoplasma capsulatum, posadasis spheriforme, Blastomyces dermatitidis, chlamydia trachomatis or Candida albicans.In other embodiments, communicable disease is caused by parasite, and such as plasmodium falciparum and toxoplasma gondii cause.

In some embodiments, cancer is noumenal tumour or hematogenous cancer.In specific examples, noumenal tumour is sarcoma or cancer, such as: fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, or other sarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdosarcoma, colorectal carcinoma, lymphoid malignancies, carcinoma of the pancreas, mammary cancer, lung cancer, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, rodent cancer, gland cancer, syringocarcinoma, sebaceous carcinoma, papillary carcinoma, papillary carcinoma, medullary carcinoma, bronchiogenic cancer, renal cell carcinoma, liver cancer, cholangiocarcinoma, choriocarcinoma, Wei Ermusishi tumour, cervical cancer, carcinoma of testis, bladder cancer, or central nerve neuroma is (as neurospongioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic tumor, oligodendroglioma, meningioma, melanoma, neuroblastoma or retinoblastoma).

In some instances, haematogenous cancer is leukemia, such as: acute leukemia (as acute lymphoblastic leukemia, acute myeloblastic leukemia, acute myelogenous leukemia and myeloblastosis, promyelocytic leukemia, myelomonocytic leukemias, monocytic leukemia and erythroleukemia); Chronic leukemia (as chronic myeloid (granulocytic) leukemia, chronic lymphocytic leukemia and lymphocytic leukemia); Polycythemia vera; Lymphoma; Lymphogranulomatosis; Non Hodgkin lymphom (painless form and advanced form); Multiple myeloma; Wal Dan Sitelunshi macroglobulinemia; Heavy chain disease; Myelodysplastic syndrome; Hairy cell leukemia; Or osteomyelodysplasia.

In some embodiments, CH2 or CH3 structural domain molecular specificity is in conjunction with tumour antigen.Tumour antigen is well known in the art and comprises such as: carcinomebryonic antigen (CEA), β-Human chorionic urgees sexual hormoue (β-HCG), α alpha-fetoprotein (AFP), the reactive AFP (AFP-L3) of lectin, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase (hTERT), RU1, RU2 (AS), intestines Carboxylesterase, mut hsp70-2, M-CSF, prostate gland enzyme (prostase), prostate specific antigen (PSA), PAP, NY-ESO-1, LAGE-1a, p53, prostein, PSMA, Her2/neu, Survivin and Telomere terminal transferase, prostate cancer antigen-1 (PCTA-1), melanic related antigen (MAGE), ELF2M, NE, ephrinB2 and CD22.CH2 or CH3 structural domain molecule can also in conjunction with any cancer-associated proteins, as IGF-I, IGF-II, IGR-IR or mesothelin (mesothelin).Other tumor associated antigen provides in following table 3.

Table 3: exemplary oncologic and tumour antigen thereof

In some embodiments, autoimmune disease is rheumatoid arthritis, the few joint type sacroiliitis of juvenile form, Collagen-induced Arthritis, Adjuvant Induced Arthritis, sjogren syndrome, multiple sclerosis, Experimental Autoimmune encephalomyelitis, inflammatory bowel (such as Crohn's disease, ulcerative colitis), autoimmunity gastratrophy, pemphigus vulgaris, psoriasis, vitiligo, type 1 diabetes, non-obese patients with type Ⅰ DM, myasthenia gravis, Grave is sick, struma lymphomatosa, sclerosing cholangitis, hardening sialadenitis, systemic lupus erythematous, autoimmune thrombocytopenic purpura, Goodpasture syndrome, Addison is sick, Systemic sclerosis, polymyositis, dermatomyositis, autoimmune hemolytic anemia or pernicious anemia.

The extensive effectiveness of CH2 and CH3 structural domain molecule at least partly on be because their sizes are little, this allows effective infiltrating tissues, comprise noumenal tumour and lymphoid tissue (wherein HIV copies), and allow effectively and tachytely with avoid the virus (such as HIV) of immunoglobulin (Ig) neutralization that produces by host immune system.Because through engineering approaches CH2 or CH3 structural domain molecule are easy to produce the high-affinity binding antibody for object antigen, so they are also useful for treatment.In addition, as described herein, CH2 or CH3 structural domain molecule can also comprise the effector molecule (such as such as, medicine, enzyme or toxin) with treatment characteristic.

As described herein, CH2 or CH3 structural domain molecule can by through engineering approaches to comprise the one or more CDR from having specific antibody to pathogenic agent (such as HIV).X5 has specific neutralizing antibody (the people .Proc.Natl.Acad.Sci.U.S.A.99:6913-6918 such as Moulard, 2002) to HIV-1.When being converted to scFv antibody from complete immunoglobulin (Ig) (IgG1) or Fab, the Neutralization effect of X5 has shown and has been increased significantly, the variable domains of described scFv antibody only containing heavy chain and light chain people .J.Virol.77:10557-10565 such as (, 2003) Labrijn.Believe that this effect is due to the limitation of size entering X5 epi-position.CH2 and CH3 structural domain molecule is less than scFv antibody, produces following hypothesis: through engineering approaches CH2 structural domain molecule (comprising one or more X5CDR) will have the neutralising capacity of enhancing because it enters the ability of epi-position.

CH2 and CH3 structural domain molecule is administered to experimenter usually used as the composition comprising one or more pharmaceutically acceptable carriers.Examples of such carriers is by the concrete composition used and partly determined by the concrete grammar being used for using said composition.Therefore, there is the preparation of the pharmaceutical composition of the present disclosure that kind is suitable for widely.

Goods for parenteral administration comprise the solution of aseptic water or non-water, suspension and emulsion.The example of non-aqueous solvent is propylene glycol, polyoxyethylene glycol, vegetables oil (as sweet oil) and injectable organic ester (as ethyl oleate).Moisture carrier comprises water, the solution of alcohol/water, emulsion or suspension, comprises salt solution and buffer medium.Parenteral vehicles comprises sodium chloride solution, woods grignard dextrose, dextrose and sodium-chlor, Lactated Ringer's solution or fixed oil.Intravenously medium comprises fluid and nutritious supplementary, electrolyte replenisher (such as based on the electrolyte replenisher of woods grignard dextrose) etc.Sanitas and other additives also can exist, such as such as biocide, antioxidant, sequestrant and rare gas element etc.

Preparation for topical application can comprise ointment, washing lotion, emulsifiable paste, gel, drops, suppository, sprays, liquid and powder.Conventional pharmaceutical carrier, water-based, powder or oiliness substrate, thickening material etc. may be necessary or make us expecting.

Powder or particle, suspension in water or non-aqueous media or solution, capsule, sachet or tablet is comprised for Orally administered composition.Thickening material, seasonings, thinner, emulsifying agent, dispersing auxiliary or tackiness agent may be expect.

Some in described composition may be used as pharmaceutically acceptable acid salt or base addition salt, described pharmaceutically acceptable acid salt or base addition salt are by with mineral acid and organic acid reaction or by reacting with mineral alkali and organic bases and formed, described mineral acid is such as hydrochloric acid, Hydrogen bromide, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid and phosphoric acid, described organic acid is such as formic acid, acetic acid, propionic acid, oxyacetic acid, lactic acid, pyruvic acid, oxalic acid, propanedioic acid, succsinic acid, toxilic acid and fumaric acid, described mineral alkali is such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, described organic bases is such as monoalkyl, dialkyl group, the thanomin of trialkylamine and arylamines and replacement.

Use and can have been come by single dose or multiple doses.Required dosage is between subjects by difference, and this depends on the species of experimenter, age, weight, general status, the concrete hemorrhagic illness that treat or outbreak, concrete CH2 or the CH3 structural domain molecule used and method of application thereof.Suitable dosage only can use normal experiment to determine by those of ordinary skill in the art.

There is provided herein comprise the independent for the treatment of significant quantity or with the through engineering approaches CH2 of pharmaceutically acceptable carrier combinations or the pharmaceutical composition of CH3 structural domain molecule.Pharmaceutically acceptable carrier includes but not limited to salt solution, buffer saline, dextrose, water, glycerine, ethanol and their combination.Carrier and composition can be aseptic, and preparation is suitable for method of application.Composition can also containing a small amount of wetting agent or emulsifying agent or pH buffer reagent.Composition can be liquor, suspension, emulsion, tablet, pill, capsule, sustained release preparation or powder.Composition can be formulated as suppository with traditional tackiness agent together with carrier (triglyceride level).Oral preparations can comprise standard vector, such as other N.F,USP MANNITOL of pharmaceutical grade, lactose, starch, Magnesium Stearate, soluble saccharin, Mierocrystalline cellulose and magnesiumcarbonate.Any common pharmaceutical carrier can be used, such as sterile saline solution or sesame oil.Described medium can also contain conventional excipient substance, such as such as: the pharmacy acceptable salt, damping fluid, sanitas etc. of adjustment osmotic pressure.Can be physiological saline and sesame oil with other media that composition provided herein uses together with method.

VIII. the purposes of antibody constant structural domain molecule for detecting

Determine that polypeptide presence or absence method is well known in the art.Such as, specific-binding agent such as CH2 structural domain molecule can be combined with other compounds, other compounds described include but not limited to enzyme, magnetic bead, colloid magnetic bead, haptens, fluorescence dye, metallic compound, radioactive compound or medicine.CH2 or CH3 structural domain molecule can also be used in immunoassay, and described immunoassay are such as but not limited to radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), Immunohistochemistry, western blotting or immune precipitation determination.These assay methods are well known in the art (see Harlow & Lane, Antibodies, A Laboratory Manual (antibody, laboratory manual), Cold Spring Harbor Publications, the description of the immunoassay format of New York (1988)).

In one embodiment, the diagnostic kit comprising immunoassay is provided.Although the details of immunoassay can along with the specific form change adopted, but generally comprise for detecting the method for antigen in biological sample the step making this biological sample contact CH2 or CH3 structural domain molecule, described CH2 or CH3 structural domain molecule reacts with object antigen-specific under immune response condition.CH2 or CH3 structural domain molecule specific binding under immune response condition is allowed to form immunocomplex, and to detect the existence of this immunocomplex (antigen of combination) directly or indirectly.

CH2 or CH3 structural domain molecule disclosed herein can also be used for fluorescence activated cell sorting (FACS).Outside the detection level that other are more complicated, FACS measures and adopts multiple Color Channel, low angle and blunt scattering detecting channels and impedance channel, to be separated or sorting cells (see United States Patent (USP) the 5th, 061, No. 620).FACS can be used for the cell by making this cell and CH2 or the CH3 structural domain molecule contacts sorting antigen positive suitably marked.But, the other technologies of different effect can be adopted to carry out the cell colony of purification and separation expectation.The survival rate of the cell fraction that the isolation technique adopted should make reservation collect maximizes.The concrete technology adopted will depend on the efficiency of separation, the cytotoxicity of method, the equipment of the easiness of separation and speed and needs and/or technical skills certainly.

Other separable programming can comprise use the magnetic resolution of the magnetic bead of CH2 or CH3 structural domain point attached bag quilt, affinity chromatography, with CH2 or CH3 structural domain molecular bond or the cytotoxic agent be combined with complement and " elutriation " that utilize antibody or CH2 or the CH3 structural domain molecule being connected to solid substrate, or other convenient technique.Specific-binding agent allows directly to be separated with the connection of magnetic bead with other solid substrates (such as sepharose 4B, polystyrene bead, hollow-fibre membrane and plastic culture dish).Can by simply the cell that the specific-binding agent by such as CH2 or CH3 structural domain molecule combines being removed by solid support physical sepn from cell suspending liquid from this cell suspending liquid.The refined condition of hatching together with the antibody of cell and solid diffusivity or CH2 or CH3 structural domain molecule and time length will be depended on specifically for the some questions of adopted system.But the selection of felicity condition is well known in the art.

Then, can after the bonding agent of the cell and solid diffusivity that allow to express object antigen be in conjunction with enough time, with physiological buffer by unconjugated cell wash-out or wash out.Then be separated from solid phase by the combining cell of any appropriate means, and use method well known in the art quantitative, the character that described appropriate means depends primarily on solid phase and the antibody adopted or CH2 or CH3 structural domain molecule.In a concrete limiting examples, by FACS quantitatively from solid phase be separated in conjunction with cell.

As known in the art, CH2 or CH3 structural domain molecule can be combined with vitamin H or fluorescence dye, described biotoxin can remove with the avidin be combined with supporter or streptavidin, and fluorescence dye can use can carry out cellular segregation with quantitative together with FACS.

CH2 or CH3 structural domain molecule can be combined with other compounds, and other compounds described include but not limited to: enzyme, paramagnetic bead, colloid paramagnetic bead, haptens, fluorescence dye, metallic compound, radioactive compound or medicine.The enzyme that can be combined with CH2 or CH3 structural domain molecule includes but not limited to alkaline phosphatase, peroxidase, urease and beta-galactosidase enzymes.Can include but not limited to by the fluorescence dye conjugated with CH2 structural domain molecule: fluorescein isothiocyanate, tetramethyl rhodamine isothiocyanate (tetramethyl rhodamine isothiocyanate), phycoerythrin, allophycocyanin and texas Red.For can with other fluorescence dyes of antibodies see Haugland, R.P., Molecular Probes:Handbook of Fluorescent Probes and Research Chemicals (molecular probe: fluorescent probe and specializes in chemistry product handbook) (1992-1994).The metallic compound that can be combined with CH2 or CH3 structural domain molecule includes but not limited to ferritin, Radioactive colloidal gold and particularly colloidal superparamagnetic pearl.The haptens that can be combined with CH2 or CH3 structural domain molecule includes but not limited to vitamin H, digoxigenin, azolactone (oxazalone) and nitrophenols.Other reagent are well known in the art.

IX. the effector function of antibody constant structural domain molecule

Through engineering approaches CH2 or CH3 structural domain can in conjunction with Fc acceptor and/or complement associated molecules, as C1q, this allows multiple effector function, comprises the cytotoxicity (ADCC) of antibody dependent cellular mediation, CDC (CDC), phagolysis, opsonization and opsonophagocytosis.In some embodiments, CH2 or CH3 structural domain molecule as herein described comprises the binding site of one or more Fc acceptor, therefore, it is possible to make these numerator mediated multiple effector functions (see with following table 4).If effector function is less desirable, so can the one or more Fc binding site of mutagenesis to stop these functions.

Antibody-antigen complex and immune cell interaction cause reacting widely, comprise multiple effector function and immunomodulatory signals.Start these by the Fc structural domain of antibody or the combination of immunocomplex and specific cell surface receptor Fc structural domain to interact.Each member of Fc receptor family identifies the immunoglobulin (Ig) of one or more isotypes by the recognition structure territory on Fc structural domain.Fc acceptor determines (such as, the Fc acceptor of IgG is called as Fc γ R) (U.S. authorizes front publication number 2006-0134709) by it to the specificity of immunoglobulin (Ig) hypotype.

Fc acceptor is the glycoprotein found on the surface of some immune system cells, and described immune system cell comprises monocyte, scavenger cell, neutrophil(e) cell, eosinophil, mastocyte, natural killer cell, B cell and dendritic cell.Fc acceptor shows various kinds of cell expression-form and effector function (see table 4).Fc acceptor allows immunocyte and the antibodies on surface being connected to microorganism or microorganism infected cells, helps these cellular identification and eliminates microbial pathogen.Fc acceptor, at their Fc region binding antibody, is the interaction activating and have the cell of Fc acceptor.

Table 4

The cell distribution of Fc acceptor and effector function

Cytophagous activation is the most common function that Fc acceptor causes.Such as, scavenger cell starts take in and kill and wound the pathogenic agent being covered with IgG by the phagolysis after the linking of its Fc γ acceptor.Another process relating to Fc acceptor is called the cytotoxicity (ADCC) of antibody dependent cellular mediation.During ADCC, the Fc γ RIII receptor for stimulating NK cell on natural killer (NK) cell surface is so that release cells toxicity molecule carrys out the target cell that apl antibody covers from the particle of NK cell.But Fc ε RI has different functions.Fc ε RI is Fc acceptor granulocyte participating in transformation reactions and defence parasitic infection.When suitable allergic effect antigen or parasite exist, at least two in IgE molecule and their Fc acceptors in surfaces of granulocytes crosslinked will trigger this cell media of being formed of release fast from its particle.

In addition, the Fc structural domain of IgG and IgM antibody can be the component of complement activation classical pathway in conjunction with C1q, C1q.When the surface bonding of IgG or IgM antibody and pathogenic agent, C1q can in conjunction with its Fc region, and this starts complement cascade, finally cause inflammatory cell raise and to the conditioning of pathogenic agent and kill and wound.

In order to provide functionality to CH2 or CH3 structural domain molecule further, can use many means well known by persons skilled in the art that effector molecule (part of such as, treating, diagnosing or detecting) is connected to CH2 or CH3 structural domain molecule.Exemplary effector molecule includes but not limited to radio-labeling, fluorescent marker or toxin.Covalency and non-covalent connection means can be used.Program for effector molecule being connected to antibody changes according to the chemical structure of effector.Polypeptide is usually such as, containing various functional group, carboxylic acid (COOH), unhindered amina (-NH ₂) or sulfydryl (-SH) group, described functional group can be used for the functional group reactions that is applicable on antibody to cause the combination of effector molecule.Or, make antibody derive to expose or connect other reactive functionality.This derivatization may relate to the connection of any one in many linkers, such as can from Pierce Chemical Company, those linkers that Rockford, IL obtain.Joint can be used to any molecule of bound antibody and effector molecule.Joint can form covalent linkage with antibody and effector molecule.The joint be applicable to well known to a person skilled in the art and include but not limited to the carbon joint of straight or branched, heterocycle carbon joint or peptide linker.When antibody and effector molecule are polypeptide, joint can be engaged with this amino acid by composition amino acid whose side base (being such as connected with halfcystine by disulfide linkage), or engages with the α carbon of end amino acid amino and carboxyl.

In some cases, when immune conjugate arrives its target spot, expect effector molecule is dissociated from antibody.Therefore, in these cases, immune conjugate will be included in the connection can cut near target spot.Cutting joint is promoted with the enzymic activity that can be experienced near target cell interior or target spot by immune conjugate from antibody release effects thing molecule or condition.

Consider reported for by various radiodiagnosis compound, radiotherapeutic compound, mark (such as, enzyme or fluorescence molecule) medicine, toxin and other agent be connected to the large metering method of antibody, and those skilled in the art can determine the appropriate methodology given agent being linked to CH2 or CH3 structural domain molecule.

Therapeutical agent comprises effector molecule, encapsulant (such as liposome), target part and part containing pharmaceutical composition itself of various medicine (such as vinealeucoblastine(VLB), daunomycin etc.) and such as cytotoxin (Pseudomonas exotoxin of such as natural or modification or diphtheria toxin).The biological effect that concrete target molecule or target cell and expectation cause is depended in the selection of concrete therapeutical agent.Therefore, such as, what therapeutical agent can be used to cause specific target cell death has Cytotoxic effector molecule.On the contrary, if only wish to cause non-lethality biological respinse, therapeutical agent can be combined with non-lethality pharmacologic agent or the liposome containing non-lethality pharmacologic agent.

Toxin can be used together with being used as CH2 or the CH3 structural domain molecule of immunotoxin.Exemplary toxin comprises Pseudomonas exotoxin (PE), ricin, abrin, diphtheria toxin and their subunit, nucleotoxin (ribotoxin), rnase, saporin and calicheamycin and botulinum toxin A to F.These toxin are well known in the art and manyly can easily obtain (such as Sigma Chemical Company, St.Louis, MO) from commercial source.

Diphtheria toxin is separated from corynebacterium diphtheriae.Usually, the diphtheria toxin used in immunotoxin is suddenlyd change to reduce or eliminate non-specific toxicity.Since 20th century the seventies understood (Laird and Groman for people, J.Virol.19:220,1976) be called the mutant of CRM107, and be used in people's clinical trial, described mutant has enzymic activity completely but has the non-specific toxicity significantly reduced.See United States Patent (USP) the 5th, 792, No. 458 and No. the 5th, 208,021, United States Patent (USP).As used herein, refer to natural diphtheria toxin or refer to remain enzymic activity but modified to reduce the diphtheria toxin of non-specific toxicity when term " diphtheria toxin " is suitable.

Ricin is the lectin RCA60 from castor-oil plant (Ricinus communis).Term " ricin " also refers to its toxin variant.Such as, see United States Patent (USP) the 5th, 079, No. 163 and No. the 4th, 689,401, United States Patent (USP).Ricinus agglutinin (RCA) exists in two forms, and these two kinds of forms are called as RCA respectively according to the molecular weight of its about 65kD and 120kD ₆₀and RCA ₁₂₀(Nicholson & Blaustein, J.Biochim.Biophys.Acta 266:543,1972).A chain is responsible for making albumen synthesize inactivation and killer cell.B chain ricin is combined with cell surface galactose residue and promote that A chain is transported in cytosol (people such as Olsnes., Nature 249:627-631,1974 and No. the 3rd, 060,165, United States Patent (USP)).

Rnase has been combined with target molecule be used as immunotoxin (see people such as Suzuki., Nat.Biotech.17:265-70,1999).Exemplary core toxin such as α-sarcina and restrictocin are people such as such as Rathore., Gene 190:31-5,1997; And Goyal and Batra, Biochem 345Pt 2:247-54, discuss in 2000.First calicheamycin is separated from micromonospora echinospora (Micromonospora echinospora), and be cause the member of the double-strand break enediyne antitumor antibiotics family caused in the DNA of apoptosis (see such as, the people such as Lee., J.Antibiot 42:1070-87.1989).This medicine be immunotoxin in clinical trial toxin part (see such as, the people such as Gillespie., Ann Oncol 11:735-41,2000).

Abrin comprises the toxicity lectin from jequirity (Abrus precatorius).Toxic component abrin a, b, c and d have the molecular weight of about 63kD to 67kD, and polypeptide chain A and B connected by two disulfide linkage forms.A chain inhibition albumen synthesizes; B chain (abrin-b) is combined with D-galactose residue (see people such as Funatsu., Agr.Biol.Chem.52:1095,1988; And Olsnes, Methods Enzymol.50:330-335,1978).

There is provided following examples so that some concrete feature and/or embodiment to be described.These embodiments should not be interpreted as the present invention to be limited to described specific features or embodiment.

Embodiment

Embodiment 1: the generation in the library of antibody CH2 structural domain, wherein said antibody CH2 structural domain is with containing amino-acid residue random mutation being any one ring in four residues Y, S, A or D

In this embodiment, construct the CH2 structural domain of sudden change, wherein by Y, S, A or D residue of 10 random arrangements, ring 1 is added that the C of this ring holds the another one G of end to replace.Similarly, by Y, S, A or D residue of 6 random alignment, ring 3 is added that the C of this ring holds the other G of end to replace.Three steps are divided to produce DNA library.

First, use CH2DNA to produce and contain the ring 1 of sudden change and two fragments of ring 2 respectively: fragment 1 and fragment 2.Fragment 1 uses N to hold primer (5'GCA CTG GCT GGTTTC GCT ACC GT GGCC CAGGC GGCC GCA CCT GAA CTC CTG 3'; SEQ ID NO:6) and ring 1 reverse primer (5'CAC GTA CCA GTT GAA CTT GCCAKM AKM AKM AKM AKM AKM AKM AKM AKM AKM CAC CACCAC GCA TGT GAC 3'; SEQ ID NO:7) produced by pcr amplification, wherein K=G or T, and M=A or C.Fragment 2 is by using ring 1 forward primer (5'AAG TTCAAC TGG TAC GTG 3'; SEQ ID NO:8) and ring 3 reverse primer (5'GAT GGTTTT CTC GAT GGG GCC AKM AKM AKM AKM AKM AKM GTTGGA GAC CTT GCA CTT G 3'; SEQ ID NO:9) produce.

The second, by using the montage of overlap-extension PCR (SOE) PCR, these two fragments are engaged.During the second step of SOE PCR, except this N holds primer, C is also used to hold primer (5'GGT GCA GAA GAT GGT GGT GGCC GGCCT GGCC TTT GGC TTTGGA GAT GGT TTT CTC GAT G 3 '; SEQ ID NO:10) to introduce the restriction site Sfi1 needed for next cloning process at the two ends of DNA.

3rd, the sudden change CH2 fragment Sfi1 of amplification digests and is connected in the phagemid vector with identical enzymic digestion.Use Amicon Ultra-4centricon by washing three times with distilled water to connecting product desalination, then by Electroporation Transformation TG1 cell.

Below show the cloned sequence (table 5) of 20 Stochastic choice of inverting TG1 cell, show successfully to be created by four residues Y, S, D and A there is random ring 1 and the CH2 mutant of ring 3.

Table 5

There is the fragment of the sudden change CH2 sequence of random ring 1 and ring 3

(X2-22 represents the title of clone)

Ring 1

x9 PEVTCVVV YYDSAAAYAY GKFNWYVDG VEVHNAKTKPREEQYNSTYR

(SEQ ID NO:11)

x14 PEVTCVVV YYSASAAASA GKFNWYVDG VEVHNAKTKPREEQYNSTYR

(SEQ ID NO:12)

x13 PEVTCVVV YDSDYASSDD GKFNWYVDG VEVHNAKTKPRKEQYNSTYR

(SEQ ID NO:13)

x15 PEVTCVVV AYSDDAAAYD GKFNWYVDG VEVHNAKTKPREEQYNSTYR

(SEQ ID NO:14)

x10 PEVTCVVV DADDDYYYYY GKFNWYVDG VEVHNAKTKPREEQYNSTYR

(SEQ ID NO:15)

x2 PEVTCVVV DDAYYDADYYY GKFNWYVDGVEVHNAKTKP REEQYNSTYR

(SEQ ID NO:16)

x11 PEVTCVVV DAAYDYSY GKFNWYVDG VEVHNAKTKPREEQYNSTYR

(SEQ ID NO:17)

x19 PEVTCVVV DYDSDDAYAD GKFNWYVDG VEVHNAKTKPREEQYNSTYR

(SEQ ID NO:18)

x16 PEVTCVVV SYYDSDSYSA GKFNWYVDG VEVHNAKTKPREEQYNSTYR

(SEQ ID NO:19)

x4 PEVTCVVV DDAYADDASA GKFNWYVDG VEVHNAKTKPREEQYNSTYR

(SEQ ID NO:20)

x17 PEVTCVVV SYYSDSDYDD GKFNWYVDG VEVHNAKTKPREEQYNSTYR

(SEQ ID NO:21)

x12 PEVTCVVV DDDSYYSYDD GKFNWYVDG VEVHNAKTKPREEQYNSTYR

(SEQ ID NO:22)

x22 PEVTCVVV YDASDYADAY GKFNWYVDG VEVHNAKTKPREEQYNSTYR

(SEQ ID NO:23)

x8 PEVTCVVV ADAAAYAYAD GKFNWYVDGVEVHNAKTKP REEQYNSTYR

(SEQ ID NO:24)

x7 PEVTCVVV ASDSSDDYD GKFNWYVDG VEVHNAKTKPREEQYNSTYR

(SEQ ID NO:25)

x5 PEVTCVVV AAAAADADYY SKFNWYVDG VEVHNAKTKPREEQYNSTYR

(SEQ ID NO:26)

x20 PEVTCVVV YDDAAYADDY GKFNWYVDG VEVHNAKTKPREEQYNSTYR

(SEQ ID NO:27)

x21 PEVTCVVV SADASDYD GKFNWYVDG VEVHNADTKPREEQYNSTYR

(SEQ ID NO:28)

x23 PEVTCVVV DDDAADAYYY GKFNWYVDG VEVHNAKTKPREEQYNSTYR

(SEQ ID NO:29)

x3 PEVTCVVV YDSDDDYDYA GKFCWYVDG VEVHNAKTKPREEHYNSTYR

(SEQ ID NO:30)

Ring 3

x9 VVSVLTVLHQ DWLNGKEYKC KVSN AASAYSGPIEKTISKA K(SEQ ID NO:31)

x14 VVSVLTVLHQ DWLNGKEYKC KVSN ADDADAGPIEKTISKA K(SEQ ID NO:32)

x13 VVSVLTVLHQ DWLNGKEYKC KVSN AADAYAGPIEKTISKA K(SEQ ID NO:33)

x15 VVSVLTVLHQ DWLNGKEYKC KVSN AADYSDGPIEKTISKA K(SEQ ID NO:34)

x10 VVSVLTVLHQ DWLNGKEYKC KVSN AADAADGPIEKTISKA K(SEQ ID NO:35)

x2 VVSVLTVLHQ DWLNGKEYKC KVSN DASASSGPIEKTISKA K(SEQ ID NO:36)

x11 VVSVLTVLHQ DWLNGKEYKC KVSN DDYAASGPIEKTISKA K(SEQ ID NO:37)

x19 VVSVLTVLHQ DWLNGKEYKC KVSN DAYASDGPIEKTISKA K(SEQ ID NO:38)

x16 VVSVLTVLHQ DWLNGKEYKC KVSN DADDASGPIEKTISKA K(SEQ ID NO:39)

x4 VVSVLTVLHQ DWLNGKEYKC KVSN AADDDSGPIEKTISKA K(SEQ ID NO:40)

x17 VVSVLTVLHQ DWLNGKEYKC KVSN ADAYAYGPIEKTISKA K(SEQ ID NO:41)

x12 VVSVLTVLHQ DWLNGKEYKC KVSN ADDYDYGPIEKTISKA K(SEQ ID NO:42)

x22 VVSVLTVLHQ DWLNGKEYKC KVSN YSDSAAGPIEKTISKA K(SEQ ID NO:43)

x8 VVSVLTVLHQ DWLNGKEYKC KVSN YAASAYGPIEKTISKA K(SEQ ID NO:44)

x7 VVSVLTVLHQ DWLNGKEYKC KVSN YDDDADGPIEKTISKA K(SEQ ID NO:45)

x5 VVSVLTVLHQ DWLNGKEYKC KVSN YYDYDYGPIEKTISKA K(SEQ ID NO:46)

x20 VVSVLTVLHH DWMNGKEYKC EVSN DADSADGPIKKTISKA K(SEQ ID NO:47)

x21 VVSVLTVLHH DWLNGEEYKC KVSN DASDDAGPIEKTIS.A K(SEQ ID NO:48)

x23 VVSVLTVLHQ DWLNGKEYKC KVSN ADDAYAGPIEKTISKA K(SEQ ID NO:49)

x3 VVSVLTVLHH YWMNGEDYKC EVSN DSYSDDGPIKKTISKA K(SEQ ID NO:50)

Embodiment 2: the CDR3 from people's antibody is moved in CH2 support

In this embodiment, by D-loop A-B and ring E-F, the people VH CDR3 (H3) from antibody library is moved in CH2.First, use five PCR by H3 D-loop A-B.First twice PCR creates by using following primer two the CH2 fragments not having ring A-B: for fragment 1-forward 1 primer (5'TAG CGA TTC GCT ACC GTG GCCCAG GCG GCC CCT GAA CTC CTG GGG GGA CC 3'; SEQ ID NO; 51) and reverse 1 primer (5 ' TCC CCC CAG GAG TTC AGG TGC 3 '; SEQ ID NO; 52), for fragment 2-forward 2 primer (5 ' TGC GTG GTG GTG GAC GTGAGC 3 '; SEQ ID NO:53) and reverse 2 primers (5'TAG GCA TGC ATC TGCATG GTG GCC GGC CTG GCC TTT GGC TTT GGA GAT GGT TTTCTC GAT GG 3'; SEQ ID NO:54).Forward 1 primer and reverse 2 primers contain the restriction site of the N end of end product and the SfiI needed for C end.Reverse 1 primer and forward 2 primer contain the end sequence needed for SOE PCR subsequently.Third time, PCR was used as the antibody VH library of template and two kinds of mixtures of three kinds of primers, and it is designed to the different H3 that increases separately.The mixture of forward primer contains H3 forward primer 1:5 ' GAA CTC CTG GGG GGACCG GCY AYR TAT TAC TGT GYG 3 ' (SEQ ID NO:55), H3 forward primer 2:5 ' GAA CTC CTG GGG GGA CCG GCY TTR TAT TAC TGT GYG 3 ' (SEQ ID NO:56), and H3 forward primer 3:5 ' GAA CTC CTG GGG GGACCG GCY GTR TAT TAC TGT GYG 3 ' (SEQ ID NO:57).The mixture of reverse primer contains H3 reverse primer 1:5 ' GCT CAC GTC CAC CAC CAC GCAGGT GCC CTG GCC CCA 3 ' (SEQ ID NO:58), H3 reverse primer 2:5 ' GCTCAC GTC CAC CAC CAC GCA GGT GCC ACG GCC CCA 3 ' (SEQ ID NO:59), and H3 reverse primer 3:5 ' GCT CAC GTC CAC CAC CAC GCAGGT GCC AYG GCC CCA 3 ' (SEQ ID NO:60).Third time, PCR created the mixture containing the fragment with end sequence H3, and described end sequence is designed to overlap with the respective ends sequence of reverse 1 primer and forward 2 primer.By the fragment that these two CH2 fragments and the fragment containing H3 are replaced by H3 to produce wherein ring AB as the primer in SOE PCR and template.By using forward 1 primer and this fragment mixture of reverse 2 primer amplification.The fragment Sfi1 of amplification digests and is connected in the phagemid vector (pComb3X or pZUD) with identical enzymic digestion.Amicon Ultra-4 centrifugal ultrafiltration pipe is used to carry out desalination by washing three times with distilled water to connecting product, then by Electroporation Transformation TG1 cell.

Similar program can be used for D-loop E-F, except: for amplified fragments 1, use another primer-reverse primer 12 (5 ' GTA CGT GCT GTT GTA CTG CTC 3 '; SEQ ID NO:61) replace reverse primer 1; For amplified fragments 2, use another primer-forward primer 22 (5 ' AAG GTC TCC AAC AAA GCC CTC 3 '; SEQ ID NO:62) replace forward primer 2; And be different for amplification H3, H3 primer.Under these circumstances, the mixture of forward primer contains H3 forward primer 12:5 ' GAG CAG TAC AACAGC ACG TAC GCA GCY AYR TAT TAC TGT GYG 3 ' (SEQ ID NO:63), H3 forward primer 22:5 ' GAG CAG TAC AAC AGC ACG TAC GCAGCY TTR TAT TAC TGT GYG 3 ' (SEQ ID NO:64), and H3 forward primer 32:5 ' GAG CAG TAC AAC AGC ACG TAC GCA GCY GTR TAT TACTGT GYG 3 ' (SEQ ID NO:65).The mixture of reverse primer contains H3 reverse primer 12:5 ' GAG GGC TTT GTT GGA GAC CTT GGT TCC CTGGCC CCA 3 ' (SEQ ID NO:66) under these circumstances, H3 reverse primer 22:GAG GGC TTT GTTGGA GAC CTT GGT GCC ACG GCC CCA 3 ' (SEQ ID NO:67), and H3 reverse primer 32:5 ' GAG GGC TTT GTT GGA GAC CTT GGT GCCAYG GCC CCA 3 ' (SEQ ID NO:68).Finally, two ring A-B and E-F can replace with VH H3.Under these circumstances, after ring A-B is replaced by H3, in the fragment obtained, ring E-F is replaced by the H3 recombinated at random.

Shown below the sequences (table 6) of 19 clones from the TG1 cell Stochastic choice transformed with two rings of being replaced by H3, show the successful implantation of H3.Fig. 4 illustrates the protein expression of some clones in these clones.The pillar location of mutating molecule indicates with arrow.

Table 6

There is the fragment of the H3 of implantation

#2-38 (being respectively SEQ ID NO:69-87) represents the title of clone

Embodiment 3: the through engineering approaches of stable CH2 mutant and sign

In this embodiment, two CH2 mutant of the stability showing raising compared with parental wild-type CH2 are identified.Because CH2 framework is stablized by the internal disulfide bonds between chain B and chain F, so the extra disulfide linkage of hypothesis between other chains can provide the entirety of CH2 stability to improve.Several positions in chain A and chain G are suddenlyd change, and one of them creates highly stable sudden change CH2, and called after m01, wherein L is (at sequence GPSVF lin FPPKPKDTL (SEQ ID NO:88)) and first K (at sequence E kin TISKAK (SEQ ID NO:89)) be mutated into C.Another mutant, called after m02, wherein V is (at GPS vin FLFPPKPKDTL (SEQ ID NO:90)) and E kfirst K in TISKAK (SEQ ID NO:91) is mutated into C, shows stability and improve compared with parental generation CH2, but lower than the stability of m01.

Materials and methods

The clone of CH2 structural domain, expression and purification.Being cloned into by people γ 1CH2 in bacterial expression vector and being used for transform Escherichia coli strain HB2151 cell, making to grow to optical density(OD) in the SB substratum of this cell at 37 DEG C is OD ₆₀₀~ 0.6 – 0.8.12 – 16 hours are continued at 37 DEG C of abduction deliverings with 1mM IPTG.Collect bacterial cell and be resuspended in (50mM TrisCl, 450mM NaCl, pH8.0) in the buffer A of 1:10 (volume of buffer A: culture volume).Polymyxin B-sulfate USP (Sigma-Aldrich, MO) (0.5mu/ml) is added into (1:1000 Polymyxin B-sulfate USP volume: culture volume) in this suspension.Subsequently by making cell lysate clarify at 4 DEG C with the centrifugal 45min of 15,000rpm, and test its expression by SDS-PAGE and Western.By using the supernatant liquor of HiTrap Chelating HP Ni-NTA post (GE Healthcare, NJ) the purifying clarification of 1ml.With buffer B (50mM TrisCl, 450mM NaCl, 200mM imidazoles, pH 8.0) after wash-out, by Amicon Ultra – 15 centrifugal filtration equipment (MILLIPORE, MA) remove imidazoles, and the albumen of purifying is remained on buffer A or PBS (9.0g/L NaCl, 144mg/L KH ₂pO ₄, 795mg/LNa ₂hPO ₄, pH 7.4) in.Check purity of protein by SDS-PAGE and pass through to measure UV absorbancy determination protein concentration.

The design of CH2 mutant and plasmid construction.In order to design CH2 mutant, use Fc crystalline structure.By selecting five mutant V10/E103 to C10/C103, F11/K104 to C11/C104, L12/T105 to C11/C105, L12/K104 to C12/C104 and V10/K104 to C10/C104 for characterizing (the people such as Humphrey with computer program VMD 18.6 analytical structure, J Mol.Graph.14:33-38,1996).They are obtained by the site-directed mutagenesis of PCR-based and are cloned in bacterial expression vector.Confirm that these are cloned and use it for transform Escherichia coli strain HB2151 by direct Sequencing.Similarly to express and these mutant of purifying with wild-type CH2.

Size exclusion chromatography.CH2, CH2m01 and CH2m02 of purifying are loaded into and operate in to assess the formation of oligomer in Hiload26/60Superdex 75HR 10/30 post (GE Healthcare, NJ) on BASIC pH/C chromatographic system (GE Healthcare, NJ).Select buffer A as mobile phase.Use the gel filtration standards be made up of zymohexase (158kD), bovine serum albumin (67kDa), Protalbinic acid (44kDa), chymotrypsinogen A (25kD) and ribonuclease A (17kDa) to determine the molecular weight of CH2, CH2m01 and CH2m02.

By mass spectrum determination disulfide linkage.Through Voyager 4700MALDI-TOF/TOF mass spectrum (Applied Biosystems, CA), by comparing the molecular weight after alkylation at the reduction of (A) all SH group and alkylation and (B) original free SH group and disulfide linkage do not reduce, determine the sum of the disulfide linkage in CH2, CH2m01 and CH2m02 of purifying.Reduction uses TCEP to carry out, and alkylation iodo-acid amide carries out.

Circular dichroism (CD).The secondary structure of CH2, CH2m01 and CH2m02 is determined by circular dichroism (CD) spectrography.The albumen of purifying is dissolved in PBS with the final concentration of 0.49mg/ml, and at AVIV Model 202CD spectrometer (Aviv Biomedical, NJ) upper record CD spectrum.Use the colorimetric pool being used for the 0.1cm light path that natural structure measures at 25 DEG C of record wave spectrums.By the thermodynamic stability of the CD signal measurement 216nm of 1 DEG C/min heating rate in record 25-90 DEG C of temperature range.After the heating, the wave spectrum of 90 DEG C is recorded.For the assessment of refolding, all samples is remained on 4 DEG C and spend the night and again measure at 25 DEG C.By external probes sensor record temperature and the temperature-ratio being gone out microcell inside by the calibration calculations low about 2-3 of temperature DEG C (for the temperature of 20 DEG C to 80 DEG C, its scope is 1.9 DEG C to 3.8 DEG C) being measured by external sensor.

Dsc (DSC).The thermostability of CH2, CH2m01 and CH2m02 is monitored further with VP-DSC micro calorimeter (MicroCal, Northampton, MA).In PBS (pH 7.4), the concentration of three kinds of albumen is 1.5mg/ml.The heating rate adopted is 1 DEG C/min and scans from 25 DEG C to 100 DEG C.

Spectrofluorimetry.Photofluorometer Fluoromax-3 (HORIBA Jobin Yvon, NJ) have recorded the interior fluorescence of CH2, m01 and m02.Use the excitation wavelength of the protein concentration 280nm of 10 μ g/ml to carry out interior fluorescence measurement, and have recorded 25 DEG C of emission spectrums from 320nm to 370nm.Employ the buffer A that there is 0mM to 8mM urea.For all samples, the Correction of the fluorescent spectrum background fluorescence of solution (damping fluid+denaturing agent).The fluorescence intensity of 340nm is used for unfolding assessment.

Nucleus magnetic resonance (NMR).For NMR experiment, first intestinal bacteria are grown in 2 × YT.By single colony inoculation in 3mL 2 × YT about 3 hours, then check opacity and bacterium is transferred in 1 liter of 2 × YT substratum and grow until reach OD at 37 DEG C further ₆₀₀~ 0.8 – 0.9.Then centrifugal for cell culture removing 2 × YT substratum is replaced 2 × YT substratum with M9 minimum medium, M9 minimum medium respectively with ¹⁵n NH ₄cl and ¹³c glucose is as unique ¹⁵n and ¹³c originates (17).By cell 30 DEG C of overnight incubation, and induce with 1mMIPTG.The cell suspension of results is continued 1h in TES damping fluid (1L culture 10mL damping fluid) on ice.4 hours are continued, the osmotic shock of induction release periplasm protein by adding 1.5 volume TES/5 on ice.Then at 4 DEG C by supernatant liquor dialysed overnight in the dialysis buffer liquid (50mM TrisCl, 0.5M NaCl).By the method purifying protein of above-described initial purification.Then the fraction of the albumen containing significant quantity is loaded on Sephacryl S-200 post (GE Healthcare, NJ) for being further purified.To the sample collection of fraction be separated in buffer A.

Manage in the 40mM TrisCl damping fluid of the pH 7.8 in (Shigemi Inc, PA) in 5-mm Shigemi at 25 DEG C and carry out NMR experiment, this damping fluid contains at 95%H ₂o/5%D ₂the sample volume of the 64mM NaCl in O and about 300 μ l, the protein concentration had is 0.5-0.8mM.The Bruker Avance 600MHz instrument (Bruker Instruments, MA) being furnished with cryogenic probe is used to carry out NMR experiment.Water upset (water-flip back) sequence is used for ¹h- ¹⁵n HSQC and ¹h}- ¹⁵n NOE tests to make the exchange between the proton and the proton of water of acid amides minimize (Grzesiek and Bax, J.Am.Chem.Soc.115:12593,1994).For the acquisition dimension (acquisition dimension) of 8012Hz spectrum width, record has 1024 complex points (complex point) ¹h- ¹⁵n hsqc spectrum, and for indirect (t ₁) dimension, record has 256 complex points ¹h- ¹⁵n hsqc spectrum.{ ¹h}- ¹⁵n NOE experiment is undertaken by record two groups spectrum with similarity number point of destination, has respectively 3 seconds and 4 seconds duplicate delays and do not have proton saturated (Gong and Ishima, J.Biomol.NMR37:147-157,2007).The uncertainty of NOE value is estimated from the square root of the variance noise (r.m.s.d.noise) of these two spectrums and peak height.

Signal distributes (Signal assignment) and test based on HNCA, CBCACONH, CCONH of CH2 structural domain, and HNCACB and CBCACONH of CH2m01 structural domain with ¹³c, ¹⁵n assesses NOESY (people such as Kay, J.Magn.Reson.89:496-514,1990 simultaneously; Muhandiram and Kay, J.Magn.Res.Series B.103:203-216,1994).Use nmrPipe to NMR data mart modeling and analysis (people such as Delaglio., J.Biomol.NMR 6:277-293,1995; Masse and Keller, J.Magn.Reson.174:133-151,2005).For the color meaning that the chemical shift on CH2 backbone structure changes, calculate standardized chemical shift and change its mean value and standard deviation (s.d.), and be divided into four classes: δ _norm>3.0 (redness), 3.0> δ _norm>2.0 (orange), 2.0> δ _norm>1.0 (yellow) and (4) δ _norm<1 (blueness).

Result

Be separated, not glycosylated people γ 1CH2 structural domain is metastable.People γ 1 heavy chain CH2 (Fig. 5 A) is cloned in bacterial expression vector, expression and purification as described above.People γ 1CH2 take high level expression as soluble proteins (bacterial cultures more than 10mg often rises) and is high soluble (more than 10mg/ml).As by size exclusion chromatography measure, it in the PBS of pH 7.4 be monomer (Fig. 5 B) (people such as Prabakaran., Acta Crystallogr.B.64:1062-1067,2008).The SDS-PAGE of people γ 1CH2 discloses the apparent molecular weight (MW) of about 14-15kDa, and this value is close to the MW calculated (14.7kDa comprises His and FLAG label).As expected, it more much smaller than the MW of scFv, Fab and IgG1 (Fig. 5 C).

Found that the not glycosylated mouse CH2 structural domain be separated is relatively unstable (as passed through the T measured by circular dichroism (CD) under physiology relevant temperature before _m=41 DEG C (people such as Feige., J.Mol.Biol.344:107-118,2004).The sequence of people CH2 is different from the sequence of mouse CH2, and this can produce different stability (Fig. 5 A).In order to the thermodynamic stability of test person γ 1CH2, use both CD and dsc (DSC).As passed through measured by CD, the secondary structure of CH2 is made up of β chain at 25 DEG C.CH2 unfolding starts at about 42 DEG C and completes (Fig. 6 A) at about 62 DEG C, and have the Tm (Fig. 6 A) of the calculating of 54.1 ± 1.2 DEG C, unfolding is reversible (Fig. 6 A).Similar results (Tm=55.4 DEG C, Fig. 6 B) is obtained by DSC.Because this person γ 1CH2 is more stable significantly than its mouse homologue.

There is design and the generation of the through engineering approaches people γ 1CH2 structural domain of extra disulfide linkage.In order to improve the stability of people CH2 further, through engineering approaches holds A chain and C to hold extra disulfide linkage between G chain at N.Inference thinks that the degree of freedom limiting these two chains may cause the reduction of unfolding degree.At first, these mutant are designed based on the CH2 crystalline structure in complete Fc, this crystalline structure is very similar to the crystalline structure of the CH2 of the separation be recently in the news, although in some rings and end exist some difference (people such as Prabakaran., Acta Crystallogr.B.64:1062-1067,2008).Based between two C alpha-carbon of albumen with known structure distance (people such as Dani., Protein Eng.16:187-193,2003; Pellequer and Chen, Proteins 65:192-202,2006) and the direction of key, have selected the five pairs of amino acid replaced by Cys: V10/E103, F11/K104, L12/T105, L12/K104 and V10/K104 (numbering starts from 1:Ala, the numbering 231 corresponding in γ 1 heavy chain) (Fig. 5 A; SEQ ID NO:5).(L12/K104 to C12/C104, between L12 and K104 for two mutant of respectively called after m01 and m02 and V10/K104 to C10/C104, between V10 and K104 ) (Fig. 7) be high soluble and to be equivalent to or higher than the horizontal expression (Fig. 8) of CH2.

The existence of extra disulfide linkage is confirmed by mass spectrum.As expected, in CH2, the number of disulfide linkage is one, and disulfide linkage number is two (tables 7) in mutant m01 and m02.Select these mutant for further sign.

Table 7

The disulfide linkage number determined by mass spectrum

N _cys＝(M _R/A)/57

N _SH＝(M _A-M _D)/57

Number=(the N of disulfide linkage (-s-s-) _cys-N _sH)/2

M01 and m02 is more stable significantly than CH2.By the thermodynamic stability of CD and dsc measurement m01, m02 and CH2, and they are by using urea and fluorescence spectrophotometry for the stability of chemical agent.In all cases, these two mutant more stable than CH2 many (Fig. 9).The CD spectrum of CH2, m01 and m02 shows them and has high β-pleated sheet structure content (Fig. 9 A and 9B) at 25 DEG C.Along with temperature raises β-pleated sheet structure structure destroyed (Fig. 9 C) gradually.At 90 DEG C, this structure is in unfolding state (Fig. 9 A and 9B).Also as reported before, by two-state model matching sigmoid curve people l., J.Mol.Biol.344:107-118 such as (, 2004) Feige.Notably, the temperature (be respectively Tm=73.8 ± 1.7 DEG C and 65.3 ± 0.6 DEG C) that 50% unfolding of m01 and m02 occurs is significantly higher than natural CH2 (54.1 ± 1.2 DEG C) (Fig. 9 C).CH2 and m01 be refolding reversibly; But m02 only partly refolding (Fig. 9 A and Fig. 6 A is compared to Fig. 9 B).

Similar results is obtained by DSC.The melting temperature(Tm) of m01 and m02 is more much higher than the melting temperature(Tm) of natural CH2, also increases about 20 DEG C and 10 DEG C (Fig. 9 D) respectively.Interestingly, the unfolding of m02 is wider than the unfolding of CH2 and m01 and have lower peak.This phenomenon can cause by existence dimeric in m02.

The stability of the unfolding that m01 and m02 guides for chemistry is also higher than CH2 (Fig. 9 E).Urea is used to measure interior fluorescence spectrum as chemical agent.Also by two-state model matching unfolding to the dependency of urea concentration.The urea concentration (being respectively 6.8M and 5.8M) that 50% unfolding of m01 and m02 occurs is higher than CH2 (4.2M).

Observe the only monomer fraction of m01, and m02 contains a small amount of polymer substance, measuring major part by SEC is dimer (Figure 10).Because the excellent specific property of m01, select it for further sign.Be also tested for the stability of truncate CH2 and the stability of truncate m01, wherein the first seven N holds residue (the residue 1-7 of SEQ ID NO:5) deleted.These truncate protein expression go out high stability.50% unfolding temperature (Figure 10 B) of CH2 and m01 (being respectively 54 DEG C and 74 DEG C) of correspondence that 50% unfolding temperature (being respectively 62 DEG C and 79 DEG C) measured by CD is significantly higher than (respectively 8 DEG C and 5 DEG C).

The structural conservation of M01.In order to check the structural perturbation caused by cysteine mutation, solution NMR being carried out to CH2 structural domain and m01 mutant and has tested. ¹h- ¹⁵n hsqc spectrum generally shows associating and providing " fingerprint " of protein main chain between proton that nitrogen-atoms directly combines with them.Each CH2's and m01 ¹h- ¹⁵n hsqc spectrum (recording in identical experiment condition) all shows one group of peak, shows that albumen is completely folding in the solution.In the structural region of albumen, main chain ¹⁵n, C ' and C _αchemical shift in two kinds of albumen, be appointed as about 75%.In m01, the C of residue Cys _αand C _βthe chemical shift of measurement be respectively 57.6ppm and 37.7ppm, and the C of Cys104 _αand C _βchemical shift be respectively 34.2ppm and 54.5ppm.These values belong to the desired extent (Sharma and Rajarathnam, J.Biomol.NMR 18:165-171,2000) of the cysteine residues of oxidation, are presented in m01 mutant and define other disulfide linkage.

N and C _αthe comparison of overall backbone chemistry displacement be also shown in the overall similarity of protein structure between CH2 and m01.But, near residue Cys31 and Cys91 and observe the change of chemical shift near the new Cys residue 12 and 104 introduced.This is unexpected, because the new disulphide bridges introduced is by connecting contiguous β chain in identical β-pleated sheet structure and Cys31-Cys91 bridge and close natural Cys31-Cys91.In CH2m01, the new disulfide linkage introduced most possibly affects the micro of the natural disulphide bonds between Cys31 and Cys91.

The relatively high ring snappiness of CH2 and m01 and rigid frame.In order to determine whether ring has snappiness in both CH2 and m01, have recorded ¹⁵n-{ ¹h}NOE.As indicated by high NOE value (higher than 0.7), determine that framework is rigidity; Phase anti-ring is on average having more snappiness.The local dynamic effect of CH2 and m01 is suitable, shows the conformational entropy that the conformational entropy being in the m01 of native state is very similar to CH2.Most probably the essential structure of CH2 structural domain and kinetics are kept, and thermostability improves along with the introducing of cysteine mutation.The flexible raising of ring also shows that both CH2 and m01 can be used as in ring the support moving into residue or Mutated residues.

Embodiment 4: there is specific CH2 structural domain molecule to HIV

The ring that this embodiment has described based on the CH2 structural domain of human IgG1 builds the phage library of synthesis to identify specifically in conjunction with the CH2 molecule of HIV coating.

Materials and methods

Primer, peptide and albumen.The all primers used in this research are all purchased from Invitrogen (Carlsbad, CA).Biotin labeled peptide is purchased from Sigma (St.Louis, MO).Bal gp120-CD4 is provided by Tim Fouts (University of Maryland, Baltimore, MD) friendship and other gp120/140 is provided by Christopher Broder (USUHS, Bethesda, MD).SCD4 is obtained with Reagent Project by AIDS research.

Library construction.Over-lap PCR is used to introduce sudden change to produce first based on the library of CH2 to ring 1 and ring 3.N is used to hold primer ACGT gGCC CAGGC gGCCgCA CCTGAA CTC CTG (SEQ ID NO:101) and ring 1 primer CAC GTA CCA GTTGAA CTT GCC AKM AKM AKM AKM AKM AKM AKM AKM AKMAKM CAC CAC CAC GCA TGT GAC (SEQ ID NO:7) produces the N end of half CH2 containing sudden change in ring 1.Use ring 1 connects primer AAG TTC AAC TGGTAC GTG (SEQ ID NO:8) and ring 3 primer GAT GGT TTT CTC GAT GGGGCC AKM AKM AKM AKM AKM AKM GTT GGA GAC CTT GCACTT G (SEQ ID NO:9) produces in ring 3 remaining CH2 with sudden change.Then primer and C is held to hold primer ACGT with N these two fragment combination by over-lap PCR step gGCC GGCCT gGCCtTT GGC TTT GGA GAT GGT TTT CTC GAT G (SEQ ID NO:102) increases, and SfiI site (underscore marks) is incorporated into two ends of CH2 fragment.In order to produce the secondary library based on the binding substances be separated from first library, ring 2 primer GCT GAC CAC ACG GTA ADH ADH ADH GTA CTGCTC CTC CCG (SEQ ID NO:103) and above-mentioned N is used to hold primer that the sudden change of ring 2 is incorporated into one-level binding substances.Use ring 2 connects primer TAC CGT GTG GTC AGC (SEQ ID NO:104) and sudden change is incorporated into one-level binding substances by ring 3 primer (2) GGA GAT GGT TTT CTC GAT GGG ADH TGGADH ADH ADH GTT GGA GAC CTT GCA (SEQ ID NO:105).By over-lap PCR step, these two fragments are engaged, and use above-mentioned identical a pair N for increasing to hold and C end primer amplification.Make PCR fragment experience SfiI digest and be connected to carrier.The product desalination of connection is transformed into and is suitable in the Electrocompetent TG1 cell of electroporation apparatus (Bio-Rad, Hercules, CA).Phage library is prepared by the transformant obtained.

Elutriation.Bal gp120-CD4, Bal gp120 and BSA directly wrapped in PBS damping fluid by Maxisorp plate (Nunc, Denmark) at 4 DEG C, spend the night for the elutriation of plate form.By about 10 of corresponding CH2 library ¹³individual bacteriophage particles is suspended in the PBS with 2% milk powder, and is applied to in the hole of albumen bag quilt.After at room temperature 2 hours, the first round each hole is washed 5 times and one is taken turns washing 10 times subsequently, then the phage TG1 cell recovery being in exponential phase of growth.The elutriation that total five takes turns is carried out to often kind of antigen in first library.To second library based on one-level binding substances, carry out three-wheel elutriation.Then mono-clonal ELISA is used to select positive colony.To often kind of antigen selection 200 clone.Only select to show in mono-clonal ELISA that the clone of OD 405>2.0 is for the preparation of phagemid and order-checking.

The expression of CH2 and refolding.Express being used in the Cloning Transformation selected as previously discussed to coli strain HB2151.In brief, single clone to be seeded in the 2xYT supplementing 100 unit amp and 37 DEG C of cultivations under shake.Work as OD ₆₀₀when reaching 0.5, add IPTG to reach the final concentration of 1mM, and culture is continued the other 3-5 hour of shake.Then collecting cell, with PXB in PBS (Sigma, St Louis) cracking, and makes supernatant liquor experience Ni-NTA agarose beads (Qiagen, Hilden, the Germany) purifying of the solvable fraction for CH2 clone.Then precipitation is resuspended in containing 25mM Tris.HCl, in the damping fluid of pH8.0,6M urea, 0.5M NaCl, makes it experience of short duration supersound process.It is made to experience Ni-NTA agarose beads (Qiagen) purifying by collected by centrifugation supernatant liquor.The CH2 experience obtained by precipitation is made then to be filtered through the low protein binding filter (Pal, Ann Arbor, MI) of 0.2 μm for the dialyzed overnight that twice PBS changes.

ELISA。Different proteantigens is diluted in PBS damping fluid with the concentration range of 1-4 μ g/ml, and it is spent the night by 96 orifice plates at 4 DEG C of bags.Then with the damping fluid of PBS+% milk powder, plate is closed.The CH2 clonal dilutions of different concns is applied to elisa plate in identical Block buffer.Except as otherwise noted, otherwise in most of ELISA, use little mouse-anti His-HRP to detect the His label of cloning C end at each CH2.Then ABTS added to each hole and obtain OD at 5-10 afterwards minute ₄₀₅.

Gel-filtration analysis.Above analyzing purifying at the Superdex7510/300GL post (GE Healthcare, Piscataway, NJ) with PBS pre-equilibration with the sample of the CH2 albumen filtered.This post is calibrated with molecular weight standard thing.By CH2 sample with the flow velocity of 0.5ml/min from wash-out this post.

Pseudovirus neutralize titrate.Substantially carry out as previously said the preparation of HIV Env pseudotype virus and neutralization (people such as Choudhry., Virology 363:79-90,2007).

Result

Based on design and the structure in people CH2 library.Suppose that the limited mutagenesis of CH2 ring may have no significant effect the folding of many mutant and stability, and the large-scale library producing potential binding substances can be used to.First, carry out the mutagenesis of ring 1 (L1) and ring 3 (L3), because they are that (ring BC, DE and FG are called L1, L2 and L3 herein for two rings of the longest (being respectively 9 and 5 residues) in molecule same side; Two spiral AB and EF are called H1 and H2; And ring CD is called L0) (Figure 11) (people such as Radaev., J.Biol.Chem.276:16469-16477,2001).Select four often occur in CDR residue (A, Y, D and S) to replace all L1 and L3 residues immediately and add an extra residue.The C also an extra residue (G) being added to each ring holds end to increase snappiness (Figure 11).Observed before these four residues (sometimes only having two) be enough to build in different frameworks specific binding surface (people such as Fellouse., Proc.Natl.Acad.Sci.USA101:12467-12472,2004; The people such as Koide., Proc.Natl.Acad.Sci.USA104:6632-6637,2007).The theoretical diversity in this library calculated is 4 ¹⁶=4.3x10 ⁹.But due to the possible sudden change (see following) produced by PCR, diversity may significantly up to the size (5x 10 in library ¹⁰).As by as indicated in the clone of analysis 100 Stochastic choice, most of mutant (may more than 80%) has correct reading frame.

The qualification of binding substances and sequential analysis.In order to test the binding substances also selected to come in handy in library, the HIV-1 envelope glycoprotein gp120 from Bal isolate (isolate) merged with two domain C D4 (is expressed as gp120 _bal-CD4) be used as antigen.After five take turns elutriation, screen 200 clones by Phage-ELISA, and be separated 15 clones with highest signal for further sign.Three clones m1a1, m1a2 and m1a3 respectively by (among 15 sequences) 7,5 and 2 sequences arrange representative, show specific enrichment.They have the similar L1 sequence primarily of D and Y composition but their L3 is very different.The abundantest clone m1a1 and m1a2 has some changes in L1 (being respectively two F in L1 and the disappearance before G), and described change is obviously due to PCR mistake.Be presented at for the ring 1 of the clone selected by Bal gp120-CD4 and ring 3 sequence with in following table 8.These results show that the support based on CH2 can support the binding substances of the phage display with different L1 and L3; The HIV-1 specificity junction mixture of new qualification is characterized as described below further.

Table 8

The ring 1 of CH2 and ring 3 sequence

^*m1a3 ' clone has ring 1 sequence identical from m1a3 but has different ring 3 sequences

The expression of solubility nAb and the sign of combination thereof.The CH2 structural domain molecule (being called " nAb ") that great majority are expressed is found (Figure 12 A) and refolding as described above in inclusion body, and often liter of bacterial cultures on average produces 10-30mg.As passed through measured by ELISA, the nAb of purifying and elutriation antigen (gp120-CD4) specific binding, have the EC50 (Figure 12 B) of scope from 500nM (m1a1 and m1a2) to low μM (m1a3).For the nAb of purifying from supernatant liquor, obtain similar result.The display of these results m1a1, m1a2 and m1a3 remain it with the binding activities of soluble form (but not phage display form), and refolding does not affect these molecules from inclusion body.Select two clone m1a1 and m1a2 with most high-affinity for further sign.

In order to test their cross-reactive, be used alone or with the mixture of solubility CD4 in use four kinds of (Bal, JRFL, R2 and 89.6) Recombinant HIV-1 envelope glycoproteins.As shown in Figure 14, m1a1 is with different degree and all protein binding.Although m1a1 is being combined with Bal gp120 in the mixture of CD4, desired by (CD4i) antibody to CD4 induction, it is combined separately very weak with gp120, and the combination of itself and other albumen is not subject to the impact of CD4 existence significantly.It is inapparent that independent Env signal reduces, and may be because reduced a little by gp120 is coated when mixing with sCD4.Similar results is obtained to m1a2.These data presentation are CD4i by the epi-position of these antibody recognition to an isolate (Bal), but to other isolates are not then.

In order to characterize their epi-position further, test the competition of m1a1 and the CD4i antibody (scFv X5 and domain antibodies m36) to have understood.Two kinds of CD4i antibody are competed with m1a2 all significantly.Therefore, m1a1 identify by the CD4i antibody of other high efficiency crossed reactions the novel conserved epitope shared, but contrary with these antibody, it obviously depends on isolate by the interactional exposure of gp120 and CD4.

Ring 1 determines binding specificity.In order to determine that the ring from CH2 clone is contributed the difference of specific binding, create two heteroclone: m1a1CH2 and m1a2CH2.The L1 coming from m1a1 and m1a2 is implanted on CH2, has replaced original L1.As passed through measured by ELISA, these hybrid antibodies, compared to m1a1, are combined with gp120-CD4 (Figure 13 A) with avidity that is approximately identical but that slightly reduce, show that L3 is not the key combined.In order to find out this support be on the whole whether to combine needed for, discrete testing is as synthetic peptide (DYDYDSYFDFG; SEQ ID NO:109) m1a1L1.Biotin labeled peptide does not combine.Also carry out relatively small conformational change in test bracket to the impact combined by producing extra disulfide linkage between chain A and chain G.As described in embodiment 3, such S-S key significantly increases the stability of CH2, and measured by NMR, the movability of any CH2 residue of not remarkably influenced and microenvironment.The antibody m1a1ss obtained is not in conjunction with any one (Figure 13 B).These data show that the binding activities of m1a1 needs support, although and change in L3 may not affect that it is active, and change relatively little in support conformation can remove deactivation.

M1a1 and m1a2 is to the neutralization of HIV-1 pseudovirus.In order to evaluate the Neutralization effect of m1a1 and m1a2, employing clone/pseudovirus and measuring and one group of nine HIV-1 isolate.Seven in these isolates to a certain extent by one or both antibody suppressions (Figure 14 A).These two kinds of antibody differentially suppress two isolates (89.6 and IIIB), and its degree and five other isolates roughly the same (Figure 14 A).Desired by the binding affinity from their rather moderate, their effect is rather moderate compared with being used as the highly efficient depressor C34 of positive control here.These results provide the evidence of following concept: can select functional binding substances from the library based on CH2 support.

Antibody (Figure 15) is improved further by the mutagenesis of second ring and the 3rd ring.Their great majority on sds gel run (Figure 16 A) with monomer.One of mutant m1b3 is mainly monomer (Figure 16 B) in gel-filtration.They combine specifically (Figure 16 C) and neutralization HIV-1 (Figure 17) in various degree.They are also competed with scFv X5 and m36, show the high conservative region (Figure 18) of their target HIV-1gp120.

This disclosure provides the antibody constant structural domain molecule comprising at least one sudden change or at least one CDR or its function fragment.Present disclosure also provides the composition and use thereof comprising antibody constant structural domain molecule.Be apparent that the fine detail of described method can be changed or improve, and do not deviate from spirit of the present invention.Our claimed all these evolutionary approach and change programme belonging to the scope and spirit of following claim.

Claims

1. comprise a polypeptide for the immunoglobulin (Ig) CH2 structural domain of IgG, wherein said CH2 structural domain comprises about 1 to about 7 amino acid whose N and holds level with both hands and cut, and wherein said polypeptide has the molecular weight being less than about 15kD.

2. the polypeptide of claim 1, its comprise about 7 amino acid whose N hold level with both hands cut.

3. the polypeptide of claim 1 or 2, at least one of the wherein ring of (i) described CH2 structural domain is suddenlyd change; (ii) described CH2 structural domain ring region at least partially by from the complementary determining region (CDR) of heterologous immunoglobulin variable domains or the displacement of its function fragment; Or (iii) the two has concurrently, and wherein said polypeptid specificity conjugated antigen.

4. the polypeptide of claim 3, wherein:

A the ring 1 of () described CH2 structural domain is suddenlyd change;

B the ring 2 of () described CH2 structural domain is suddenlyd change;

C the ring 3 of () described CH2 structural domain is suddenlyd change;

D the ring A-B of () described CH2 structural domain is suddenlyd change;

E the ring C-D of () described CH2 structural domain is suddenlyd change;

F the ring E-F of () described CH2 structural domain is suddenlyd change;

G the ring A-B of () described CH2 structural domain is replaced by CDR; And/or

H the ring E-F of () described CH2 structural domain is replaced by CDR.

5. the polypeptide of claim 4, the described CDR wherein in (g) or (h) is CDR3.

6. the polypeptide of any one of claim 1-5, wherein said CH2 structural domain also comprise 1 to 4 amino acid whose C hold level with both hands cut.

7. the polypeptide of any one of claim 1-6, described polypeptide has the molecular weight of 12kD to 14kD.

8. the polypeptide of any one of claim 1-7, wherein said CH2 structural domain is nonglycosylated.

9. the polypeptide of any one of claim 1-7, wherein said CH2 structural domain is glycosylated.

10. a nucleic acid molecule, the polypeptide of any one of its coding claim 1-9.

11. 1 kinds of carriers, described carrier comprises the nucleic acid molecule of claim 10.

12. 1 kinds of host cells be separated, it comprises the carrier of claim 11.

13. 1 kinds of compositions, it comprises the polypeptide of any one of claim 1-9 and pharmaceutically acceptable carrier.

14. 1 kinds of compositions, it comprises the polypeptide of any one of claim 1-9 be combined with effector molecule or detectable mark.

15. 1 kinds of methods identifying the recombinant C H2 structural domain of specific binding target antigen, described method comprises:

A () is provided in the library of the bacteriophage particles of their displaying on surface restructuring IgG CH2 structural domain, wherein said CH2 structural domain comprises 7 amino acid whose N end disappearances, and wherein said CH2 structural domain has the molecular weight being less than 15KD, wherein said library is by following generation:

I () provides the library of the nucleic acid molecule of the genetic diversity colony of coding CH2 structural domain, wherein said genetic diversity colony provides in the one or more ring regions by sudden change being introduced CH2 structural domain, wherein said ring region is selected from ring 1, ring 2 and ring 3, and wherein sudden change being introduced one or more ring region comprises with A, Y, the ring residue that D or S residue random permutation is all, and hold end to insert other G residue at the C-of each ring; And

(ii) in the host cell of restructuring, express the library of described nucleic acid molecule, thus CH2 structural domain is expressed on the surface of described particle, and the nucleic acid molecule of described CH2 structural domain is encoded by the genetic stocks of described particle;

B the library of described particle is contacted the particle to select target antigen described in specific binding by () with described target antigen; And

C () clones CH2 structural domain nucleic acid molecule from the particle of expression specificity in conjunction with the CH2 structural domain of described target antigen, identify the CH2 structural domain of target antigen described in specific binding thus.

16. 1 kinds of methods preparing the library of the recombinant C H2 structural domain of IgG, described method comprises (i) and sudden change is introduced in one or more ring regions of CH2 structural domain support, wherein said ring region is selected from ring 1, ring 2 and ring 3, and wherein sudden change being introduced one or more ring region comprises with A, Y, the ring residue that D or S residue random permutation is all, and hold end to insert other G residue at the C-of each ring, or (ii) replace the described ring A-B of CH2 structural domain support or a part of ring E-F with the CDR3 from heterologous immunoglobulin weight chain variable (VH) structural domain, wherein said CH2 structural domain support also comprise 7 amino acid whose N hold level with both hands cut.

The method of 17. claims 16, wherein said CH2 structural domain support also comprise 1 to 4 amino acid whose C hold level with both hands cut.

18. 1 kinds of methods identifying the recombinant C H2 structural domain of specific binding target antigen, described method is comprised the library making to be produced by the method for claim 16 or 17 and contacts recombinant C H2 structural domain to select target antigen described in specific binding with described target antigen.